Objective: To construct the eukaryotic expression vector pmCherry-N1-hAQP4-M23of aquaporin 4 (AQP4),and to detect the expression of hAQP4-M23 protein in the Chinese hamster lung cell line V79. Methods: The hAQP4-M23 gene was amplified by PCR method,and ligated into the pmCherry-N1 after double digestion to construct the recombinant plasmid pmCherry-N1-hAQP4-M23. The plasmids were transfented into V79 cells by lipidosome,and the cells were randomly divided into control group (the V79 cells without recombinant plasmid transfection) and transfection group (the V79 cells with recombinant plasmid transfection).The expressions of hAQP4-M23 in the cells in two groups were detected by RT-PCR method and fluorescence microscope;the AQP4 antibody in serum of the neuromyelitis optica(NMO) patient was bound to the cells in two groups and the activities of hAQP4-M23 in the cells in two groups were detected by immunofluorescence and water permeability assay. Results: The sequencing results showed that the AQP4 eukaryotic expression vector was successfully constructed.The fluorescence microscope results showed that the cell membrane in transfection group clearly expressed the red fluorescence.The immunofluorescence detection results showed that the cell membrane in transfection group presented the green fluorescence,indicating that the AQP4-M23 protein of cell membrane in transfection group could be bound to the serum components of the NMO patients.Compared with control group,the activity of hAQP4-M23 in the cells in transfection group was increased significantly (P<0.05). Conclusion: The eukaryotic expression vector expressing AQP4 gene is successfully constructed and the AQP4-M23 protein is successfully expressed in the V79 cell line.

Objective: To observe the improvement effect of 17β-estradiol on the intestinal mucosal mesenteric microcirculation after hemorrhagic shock (HS) in the rabbits,and to explore the relevant mechanism. Methods: Thirty-two rabbits were randomly divided into model group,saline treatment group,anisodamine treatment group and 17β-estradiol prevention group,and there were 8 rabbits in each group.The HS model was made by Chaudry method,and the changes of intestinal mucosal microcirculation were recorded;the pathomorphology of intestinal mucosa of the rabbits in various groups were observed under microscope and the Chiu's score was used to evaluate.the positive expression rates of interleukin-6 (IL-6) and CD68 macrophages in intestinal mucosa of the rabbits in various groups were detected by immunohistochemistry;the levels of 17β-estradiol,lactic acid and IL-6 in serum of the rabbits in various groups were determined by ELISA method. Results: Two hours after shock treatment,the mesenteric blood flow state of the rabbits in anisodamine treatment group and 17β-estradiol prevention group was better than that in saline treatment group (P<0.05).In model group,the mucosal glands were atrophied,the villous columnar epithelial cells were powdered and corroded; in saline treatment group,the intestinal mucosal epithelium was partially exfoliated and broken,the villi structure was incomplete,and there were many exfoliated mucosal tissues in intestinal cavity; in anisodamine treatment group,the glands were slightly atrophied,the villi were slightly edema,and the lamina propria was slightly separated;in 17β-estradiol prevention group,the intestinal mucosal glands were normal,and the villi and the lamina propria were edema. The Chiu's score of the rabbits in model group was higher than those in other three groups (P<0.05);the Chiu's score of the rabbits in saline treatment group was higher than those in 17β-estradiol prevention group and anisodamine treatment group (P<0.05).The positive expression rates of IL-6 and CD68 macrophages in intestinal mucosa of the rabbits in anisodamine treatment group and 17β-estradiol prevention group were lower than those in saline treatment group and model group (P<0.05). The level of serum 17β-estradiol of the rabbits in 17β-estradiol prevention group was significantly higher than those in the other groups (P<0.05); the levels of serum lactic acid and IL-6 of the rabbits in 17β-estradiol prevention group were lower than those in the other groups (P<0.05);the levels of serum lactic acid and IL-6 of the rabbits in anisodamine treatment group and 17β-estradiol prevention group were lower than those in saline treatment group (P<0.05). Conclusion: 17β-estradiol can improve the intestinal mucosal microcirculation injury in the HS rabbits,and its mechanism may be realized by reducing the production of lactic acid,reducing the infiltration of macrophages, and inhibiting the release of inflammatory factor IL-6.

Objective: To investigate the pathogenesis of brain injury induced by hyperoxia in the newborn rats,to elucidate the mechanism of the protect effect of prostaglandin E1 (PGE-1) through endoplasmic reticulum stress(ERS) pathway,and to provide the relevant theoretical basis for the treatment of brain injury of the newborns induced by hyperoxia. Methods: Ninety newborn Wistar rats were randomly divided into control group,hyperoxia group and hyperoxia+PGE-1 group,and there were 30 rats in each group.The rats in hyperoxia group and hyperoxia+PGE-1 group were placed in the hyperoxia box; the rats in control group were placed under the same room pressure conditions.From the 1st day of modeling,the PGE-1 was intraperitoneally injected into the rats in hyperoxia+PGE-1 group.The rats in the other two groups were injected with the same dose of normal saline.On the 1st,3rd and 7th days of hyperoxia exposure,10 rats in each group were randomly selected to detect the body weights and water contents of brain tissue;the morphology of brain tissue of the rats in three groups was observed by HE staining;the apoptosis of brain cells of the rats in three groups was observed by TUNEL staining,and the apopotic indexes were caculated;the expression amount of c-Jun N-terminal protein kinase(JNK) and phosphorylated JNK(p-JNK) proteins in brain tissue of the rats in three groups were detected by Western blotting method. Results: On the 1st,3rd and 7th days,the body weights of the rats in hyperoxia group were significantly lower than those in control group (P<0.05);the water contents in brain tissue of the rats in hyperoxia group were significantly higher than those in control group (P<0.05);compared with hyperoxia group,the body weights of the rats in hyperoxia+PGE-1 group were increased (P<0.05), and the water contents in brain tissue were decreased (P<0.05). The HE staining results showed that the cerebral cortex cells of the rats in control group were regular in shape and arranged in order,with little infiltration of inflammatory cells and edema of cells;the cortical cells of the rats in hyperoxia group were disordered,and the inflammatory cells were found in the brain parenchyma;the cerebral cortex cells of the rats in hyperoxia+PGE-1 group were arranged neatly,the cell morphology was relatively regular,the number of inflammatory cells in brain parenchyma were reduced,and the degree of brain parenchyma edema was less than that in hyperoxic group.The TUNEL staining results showed that the cells in brain tissue of the rats in control group were mainly cerebral cortical cells,and the apoptotic cells were rare and the nucleus showed homogeneous blue;in hyperoxia group,the nucleus was bright white,which were positive apoptotic cells.Compared with hyperoxia group,the number of apoptotic cells in hyperoxia+PGE-1 group was decreased significantly at the same time point.The apoptotic index in brain cells of the rats in hyperoxia group was higher than those in hyperoxia+PGE-1 group and control group (P<0.05).The expression amounts of JNK and p-JNK proteins in brain tissue of the rats in hyperoxia group were significantly higher than those in control group (P<0.05),and the expression amounts of JNK and p-JNK proteins in brain tissue of the rats in hyperoxia+PGE-1 group were significantly lower than those in hyperoxia group (P<0.05). Conclusion: The pathogenesis of brain injury induced by hyperoxia in the newborn rats may be related to the down-regulation of the expressions of ESR-related p-JNK and JNK proteins,and PGE-1 has the protective effect on it.

Objective: To study the neuroprotective effect of emodin in the model rats with ischemic stroke and its effect the on extracellular regulated protein kinase1/2 (ERK1/2) signaling pathway, and to explore the mechanism of protective effect of emodin on the ischemic stroke. Methods: A total of 200 rats were randomly divided into sham operation group (n=60 rats), model group (n=70) and emodin group (n=70). The rat models of ischemic stroke were established in model group and emodin group. The rats in emodin group were injected intraperitoneally with emodin 30 min before modeling. The neurological dysfunction scores, cerebral infarction volumes and water contents in brain tissue of the rats in various groups were measured; immunohistochemical staining was used to determine the apoptotic rates of ischemic lateral cortex cells of the rats in various groups;Western blotting method was used to determine the expression levels of Cleaved caspase-3, Bcl-2 related X protein (Bax), B lymphocyte tumor-2 (Bcl-2), ERK1/2 and phosphorylation-ERK1/2 (p-ERK1/2) proteins of the rats in various groups. Results: The rats in sham operation group had no neurological dysfunction and cerebral infarction. The neurological dysfunction score and cerebral infarction volume of the rats in emodin group were significantly lower than those in model group (t=8.331, t=3.538, P<0.05).Compared with model group, the water content in brain tissue of the rats in emodin group was decreased (t=9.507, P<0.05), the apoptotic rate of cerebral cortex cells was decreased (t=57.593,P<0.05), the expression levels of Cleaved caspase-3, Bax, and p-ERK1/2 proteins in ischemic brain tissue were decreased (t=4.088, t=4.463, t=10.659, P<0.05), and the expression level of Bcl-2 protein in ischemic brain tissue was increased (t=5.035, P<0.05). Conclusion: Emodin may inhibit the neuronal apoptosis by inhibiting the ERK1/2 signaling pathway to exert the neuroprotective effect on the rats with ischemic stroke.

Objective: To investigate the effects of ascorbic acid-polyethyleneimine carbon dots(CDs) on the proliferation, apoptosis and oxidative stress of the human osteosarcoma cell line MG63 cells, and to elucidate the biological characteristics of ascorbic acid-polyethyleneimine CDs. Methods: The ascorbic acid-polyethyleneimine CDs were synthesized with ascorbic acid and polyethyleneimine by microwave method. The proliferation rate of MG63 cells was detected by MTT method, and the percentages of MG63 cells at different cell cycles were detected by flow cytometry.The MG63 cells were divided into blank control group,negative control transfection group, GOLPH3-siRNA group, CDs group,negative control transfection+CDs group, GOLPH3-siRNA+CDs group. The expression levels of Golgi phosphorylation protein 3 (GOLPH3) gene in the MG63 cells in various groups were detected by PT-PCR method and the gene silencing efficiency was calculated; the apoptotic rates and reactive oxygen specie(ROS)levels in the MG63 cells in various groups were detected by flow cytometry. Results: Compared with blank control group, the proliferation rates of the MG63 cells were decreased with the increasing of CDs concentration; at 24 and 48 h, when the CDs concentration was more than 40 mg·L^-1, the proliferation rates of the MG63 cells were decreased significantly (P<0.05); at 72 h, when the CDs concentration was more than 20 mg·L^-1, the proliferation rates of the MG63 cells were decreased significantly (P<0.05). Compared with control group, the percentage of the MG63 cells at G₀/G₁ phase in CDs group was increased significantly(P<0.01), the percentage of the MG63 cells at S phase in CDs group was decreased significantly(P<0.01). The photoluminescence properties of CDs in the MG63 cells were seen under inverted fluorescence microscope; compared with non-CDs groups (blank control group, negative control transfection group, GOLPH3-siRNA group),the apoptotic rates of MG63 cells and the ROS levels in the MG63 cells in CDs groups(CDs group,negative control transfection+CDs group, GOLPH3-siRNA+CDs group)were increased(P<0.05). Conclusion: CDs have low toxicity and photoluminescence properties,which can arrest the cell cycle and promote the apoptosis and ROS production.GOLPH3 has the anti-apoptosis effect and inhibitory effect on the production of ROS to protect the survival of cells.

Objective: To construct the RHBDF2-shRNA lentiviral vector and to establish a stably transfected Neuro-2a-RHBDF2-shRNA cell line,and to detect the expression levels of RHBDF2 mRNA and protein in the stably transfected cell line. Methods: According to the RHBDF2 sequence in NCBI,a shRNA-oligo targeting RHBDF2 gene was designed and synthesized.It was cloned into the lentiviral vector FV067-RNAi-EGFP-Puro digested by EcoRⅠand AgeⅠrestriction endonuclease to construct the FV067-RHBDF2-shRNA lentiviral plasmid.The positive clones were screen by PCR and verified by sequencing.The HEK293T cells and Neuro-2a cells were used as the experimental materials.The Neuro-2a-FV067 Vector was used as control group,and the Neuro-2a-FV067-RHBDF2-shRNA was used as experiment group.The FV067 Vector and FV067-RHBDF2-shRNA plasmids were co-transfected into the HEK293T cells with lentiviral-assisted plasmids psPAX2 and pMD2G using Lipofectamine 2000,respectively.The lentivirus was harvested and purified 48 h after transfection.The FV067 Vector lentivirus in control group and the FV067-RHBDF2-shRNA lentivirus in experiment group were used to infect the Neuro-2a cells when the multiplicity of infection(MOI) was 50,respectively,and the expression of green fluorescence was detected under fluorescence microscope.Then 2 mg·L^-1 puromycin was used to screen the cell line stably silencing RHBDF2.Real-time fluorescence quantitative PCR was used to detect the expression levels of RHBDF2 mRNA in the cells in two groups and Western blotting method was used to detect the expression levels of RHBDF2 protein in the cells in two groups. Results: The PCR and the sequencing analysis results indicated that the interference sequence of RHBDF2-shRNA lentiviral vector was identical with the sequence of designed and synthesized RHBDF2-shRNA-oligo.The titer of FV067 Vector lentivirus was 5×10⁸ TU·mL^-1 and the titer of FV067-RHBDF2-shRNA lentivirus was 3×10⁸ TU·mL^-1.The real-time fluorescence quantitative PCR results revealed that the expression level of RHBDF2 mRNA in the stably transfencted cell line in experiment group was significantly decreased by 52.26% compared with control group(t=11.44,P=0.007 6).The Western blotting results indicated that the expression level of RHBDF2 protein in the stably transfected cell line was significantly decreased by 47% compared with control group(t=6.374,P=0.007 8). Conclusion: The FV067-RHBDF2-shRNA lentiviral vector is constructed successfully and the Neuro-2a-RHBDF2-shRNA cell line is established,and the expression levels of RHBDF2 mRNA and protein in the Neuro-2a-RHBDF2-shRNA stably transfected cell line is decreased significantly.

Objective: To construct the apoptosis model of islet INS-1 cells of the rats induced by dexamethasone(Dex),and to investigate the effect of lithium chloride(LiCl) on the apoptosis of the islet β cells induced by Dex and its possible mechanism. Methods: The INS-1 cells were divided into control group,0.1 μmol·L^-1 Dex group and LiCl+0.1 μmol·L^-1 Dex group. TUNEL staining and Annexin Ⅴ/PI staining methods were used to detect the apoptotic rates of the INS-1 cells in various groups;Real-time PCR method was used to detect the expression levels of superoxide dismutase (SOD),inducible-nitric oxidesynthase (iNOS),NADPH oxidase 4 (Nox4),NADPH oxidase (p47phox),and glycogen-synthase kinase-3β (GSK3β) mRNA in the INS-1 cells in various groups;Western blotting method was used to detect the expression levels of GSK-3β,p-GSK-3β,SOD,iNOS and Nox4 proteins in the INS-1 cells in various groups;GENMED Kit was used to detect the levels of reactive oxygen species (ROS) in the INS-1 cells in various groups,and Griess method was used to detect the levels of nitric oxide (NO) in the INS-1 cells in various groups. Results: Compared with control group,the apoptotic rate of INS-1 cells in 0.1 μmol·L^-1 Dex group was significantly increased(P<0.05),the expression levels of SOD mRNA and p-GSK-3β proteins were decreased(P<0.05),the expression levels of Nox4,p47phox,iNOS mRNA were increased(P<0.05),and the expression levels of ROS and NO in the INS-1 cells were significantly increased (P<0.05); compared with 0.1 μmol·L^-1 Dex group,the apoptotic rate of INS-1 cells in LiCl+0.1 μmol·L^-1 Dex group was significantly decreased(P<0.05),the expression levels of SOD mRNA and p-GSK-3β protein were increased(P<0.05),the expression levels of Nox4,p47phox and iNOS mRNA were decreased(P<0.05 or P<0.01),and the levels of ROS and NO were decreased (P<0.05). Conclusion: Dex can induce the apoptosis of the islet β cells of the rats.LiCl can attenuate the apoptosis of dexamethasone-induced islet β cells by inhibiting the activity of GSK-3β.

Objective: To investigate the expression of filamin A (FLNa) in cervical cancer tissue and its relationships with the clinicopathological characteristics of the cervical cancer patients, to analyze the correlations between the expression of FLNa and the prognostic indicators, to clarify the feasibility of FLNa as a prognostic indicator for cervical cancer,and to provide the evidence for early clinical evaluation on the prognosis of cervical cancer. Methods: The positive expression rates of FLNa in 55 cases of cervical cancer tissue and adjacent tissue were detected by immunohistochemical method,the expression levels of FLNa mRNA in 20 cases of cervical cancer and adjacent tissues were detected by RT-PCR method, and the relationships between the expression of FLNa and the clinicopathological parameters of the patients with cervical cancer were analyzed. Results: The immunohistochemical results showed that FLNa was localized in the cytoplasm,the positive expression rate of FLNa in the basal cells was high and it expressed partly in the interstitium.FLNa highly expressed in the cervical cancer tissue and did not express in the adjacent normal tissue.The positive expression rate of FLNa in the patients with stage Ⅰ+Ⅱ cervical cancer was lower than that in the patients with stage Ⅲ+Ⅳ cervical cancer (P<0.05).The expression of FLNa in cancer tissue of the patients with cervical cancer was associated with TNM stage,lymph node metastasis and parametrial invasion (P<0.05).The RT-PCR results showed that the expression level of FLNa in 20 cases of cervical cancer tissue was higher than that in adjacent normal tissue (P<0.05). Conclusion: FLNa may become a indicator of evaluation on the prognosis of the patients with cervical cancer.

Objective: To observe the protective effect of procyanidin B1 (PB1) on the lipopolysaccharide (LPS)-induced injury of macrophages RAW264.7, and to explore its possible mechanism. Methods: The macrophages RAW264.7 in logarithmic phase cultured in vitro were divided into control group (without treatment), LPS group (treated with 2 mg·L^-1 LPS), PB1 group (treated with 10 μmol·L^-1 PB1) and PB1 + LPS group (treated with 2 mg·L^-1 LPS and 10 μmol·L^-1 PB1). The morphology of cells was observed under microscope, the levels of reactive oxygen species (ROS) in the cells,the apoptotic rates of the cells and the expression levels of CD16/32, CD40, CD86, and Toll-like receptor 4(TLR4) on cell surface of the cells in various groups were detected by flow cytometry. Results: Compared with control group, the cells in LPS group were shrunk and round,but the morphological changes of the cells in PB1 group and PB1 + LPS group were not significant. Compared with control group, the ROS level in the cells in LPS group was significantly increased (P<0.05); compared with LPS group, the ROS level in the cells in PB1 + LPS group was significantly decreased (P<0.05). Compared with control group, the apoptotic rate of the cells in LPS group was decreased (P<0.05); compared with LPS group, the apoptotic rate of the cells in PB1 + LPS group was increased(P<0.05). Compared with control group, the expression levels of cell surface molecules CD16/32, CD40, CD86, and TLR4 in the cells in LPS group were increased (P<0.05); compared with LPS group, the expression levels of cell surface molecules CD16/32, CD40, CD86, and TLR4 in the cells in PB1 + LPS group were decreased (P<0.05). Conclusion: LPS can induce the cell injury by increasing the ROS level, and PB1 can protect the cells by decreasing the ROS level and down-regulating the expression levels of cell surface molecules CD16/32, CD40, CD86, and TLR4.

Objective: To investigate the protective effect of ginseng extract on the lipotoxic cardiomyocyte injury induced by palmitic acid (PA),and to clarify its possible mechanism. Methods: The H9c2 cells were cultured in vitro.The levels of lipid droplets in H9c2 cells after treated with different concentrations(100,200,and 1 000 μmol·L^-1) of PA were detected by oil red O staining,the survival rates of H9c2 cells after treated with different concentrations(200,400,600,and 800 μmol·L^-1) of PA were detected by MTT assay,and the apoptotic rates of H9c2 cells after treated with different concentrations(200,400,600,and 800 μmol·L^-1) of PA were detected by flow cytometry.The concentration of PA and the action time were screened;according to the screening results,the PA concentrations were selected as 100 and 200 μmol·L^-1,and the action time was 24 h.The H9c2 cells were randomly divided into control group,PA group (100 or 200 μmol·L^-1) and and different concentrations (0.2,2.0,and 20.0 mg·L^-1)of ginseng extract groups.Flow cytometry and FITC-Annexin Ⅴ/PI double staining were used to detect the apoptotic rates of the H9c2 cells in various groups,JC-1 staining was used to detect the levels of mitochondrial membrane potential (MMP) in the H9c2 cells in various groups,and DCFH-DA staining was used to detect the levels of intracellular reactive oxygen species (ROS) in the H9c2 cells in various groups. Results: Compared with control group,the levels of lipid droplets in the H9c2 cells in 100,200, and 1 000 μmol·L^-1 PA groups were increased significantly (P<0.01).Compared with control group,the survival rates of the H9c2 cells in 200,400,600,and 800 mmol·L^-1 PA groups were decreased (P<0.01),and the apoptotic rates of the H9c2 cells in 200,400,600,and 800 mmol·L^-1 PA groups were increased(P<0.01).After treated with different concentrations of ginseng extract for 24 h,compared with 100 or 200 μmol·L^-1 PA groups,the lipid levels of the H9c2 cells in 2.0 and 20.0 mg·L^-1 ginseng extract groups were decreased(P<0.01),the apoptotic rates were decreased(P<0.01),and the levels of ROS were decreased (P<0.01); the levels of MMP in the H9c2 cells in different concentrations of ginseng extract groups were decreased (P<0.01). Conclusion: Ginseng extract can inhibit the apoptosis of the cardiomyocytes induced by PA,and its mechanism may be related to the down-regulation of ROS and MMP levels in the H9c2 cells.

Objective: To explore the protective effect of nutrient complex on the rats with alcoholic liver disease(ALD),and to elucidate its mechanism. Methods: A total of 60 male Wistar rats were randomly divided into blank control group,model control group,low,medium and high doses (2.5,5.0,and 15.0 mL·kg^-1)of nutrient complex groups,and there were twelve rats in each group. The rats in blank control and model control groups were fed with distilled water and the rats in other groups were fed with different doses (2.5,5.0,and 15.0 mL·kg^-1)of nutrient complexes. The rats in blank control group were fed with distilled water again and the rats in other groups were fed with 30% wine 2 h later. The rats in various groups were administrated by gavage twice a day for 36 d.The liver tissue of the rats in various groups were taken and the levels of malonaldehyde(MDA),reduced glutathione(GSH) and triglyceride(TG) in liver tissue of the rats in various groups were detected.The pathological morphology of liver tissue of the rats in various groups was observed, Sudan Ⅲ staining was used to observe the number of lipid droplets in liver tissue and liver cells and the distribution of hepatic fat of the rats in various groups,and the steatosis of liver tissue of the rats in various groups was scored. Results: Compared with blank control group,the levels of MDA and TG in liver tissue of the rats in model control group were significantly increased(P<0.01),and the level of GSH was significantly decreased(P<0.05); compared with model control group,the levels of MDA and TG in liver tissue of the rats in low,medium and high doses of nutrient complex groups were decreased significantly(P<0.01),and the levels of GSH in liver tissue of the rats in medium and high doses of nutrient complex groups were significantly increased(P<0.05 or P<0.01). Large amounts of lipid droplets were found in the liver tissue and liver cells of the rats in model control group,the nucleus was blue and presented the pathological changes of steatosis of liver tissue. Compared with blank control group,the number of lipid droplets in liver tissue and liver cells of the rats in model control group was increased,and the pathological score of steatosis of liver tissue was increased(P<0.05); compared with model control group,the number of lipid droplets in liver tissue and liver cells of the rats in medium and high doses of nutrient complex groups was significantly decreased,and the pathological scores of steatosis of liver tissue were decreased (P<0.05). Conclusion: Nutrient complexes play a protective role in ALD by increasing the levels of antioxidants and reducing the peroxidation products in the liver tissue.

Objective: To observe the expression of forkhead box protein 3(FOXP3) in human lung adenocarcinoma tissue and the effects of FOXP3 on the cell proliferation and the chemosensitivity to cisplatin in the lung adenocarcinoma cells,and to elucidate the relevant mechanism of the resistance of lung adenocarcinoma cells to cisplatin. Methods: A total of 50 paraffin-embedded samples of cancer tissue of the patients with lung adenocarcinoma and 10 normal lung tissue samples were selected. The expressions of FOXP3 and Ki-67 in the lung adenocarcinoma and normal lung tissues were detected by immunohistochemistry,the difference in the positive expression rates of FOXP3 between lung adenocarcinoma and normal lung tissues was analyzed,and the correlation between the expressions of FOXP3 and Ki-67 was analyzed by Pearson correlation analysis.The human lung adenocarcinoma A549 cells at the logarithmic phase were transfected with siRNAs,and the A549 cells were divided into Mock group (transfected with liposome),Control-siRNA group (transfected with Control-siRNA) and FOXP3-siRNA group (transfected with FOXP3-siRNA).RT-qPCR and Western blotting methods were used to detect the expression levels of FOXP3 mRNA and protein in the A549 cells in various groups,and CCK-8 method was used to detect the proliferation activities of cells in various groups and the inhibitory rates of proliferation of the A549 cells in various groups after treated with 0,0.63,1.25,and 2.50 mg·L^-1 cisplatin;RT-qPCR and Western blotting methods were used to detect the expression levels of FOXP3 mRNA and protein in the A549 cells after treated with cisplatin. Results: The positive expression rates of FOXP3 in lung adenocarcinoma tissue and normal lung tissue were 62.0% and 13.3%,and the difference was statistically significant(P<0.05);the expression of FOXP3 in lung adenocarcinoma tissue was positively correlated with the expression of Ki-67 in lung adenocarcinoma tissue (r=0.370,P<0.01). The expression levels of FOXP3 mRNA and protein in the A549 cells in FOXP3-siRNA group were significantly decreased compared with Mock group and Control-siRNA group (P<0.01);the proliferation activities of A549 cells in FOXP3-siRNA group were significantly decreased compared with Mock group and Control-siRNA group after treated with cisplatin for 48 and 72 h (P<0.05);the inhibitory rates of proliferation of A549 cells in FOXP3-siRNA group were significantly higher than those in Mock group and Control-siRNA group after treatment with 1.25,2.50 and 5.00 mg·L^-1 cisplatin (P<0.05).The expression levels of FOXP3 mRNA and protein in the A549 cells in 1.25 and 2.50 mg·L^-1 cisplatin groups were both higher than those in 0 mg·L^-1 cisplatin group (P<0.05). Conclusion: Silencing FOXP3 can inhibit the levels proliferation and increase the chemosensitivity to cisplatin of the lung adenocarcinoma cells.

Objective: To investigate the preparation process of gene chips for individualized treatment of thrombotic diseases (TD),and to elucidate a rapid and high-throughput method for detecting the drug metabolism genes of clopidogrel and warfarin. Methods: Different kinds of probes and primers were designed according to the clopidogrel drug metabolism gene sites (CYP3A4,CYP2C19*17,CYP2C19*2, and CYP2C19*3) and warfarin drug metabolism gene sites (CYP4F2*3,GGCX,VKORC1-2,VKORC1-1,CYP2C9*2, and CYP2C9*3) to prepare the gene chip detection kit for individualized treatment of TD.The steps of microarray detection were DNA extraction,PCR amplification,hybridization,elution and scanning.The sensitivity,repeatability and stability of the gene chips were evaluated. The samples of 150 subjects with TD from three regions were selected,and the drug metabolism genes were detected.Based on the medical guidances in the clinic and the disease recovery status after 6 months,the effectiveness of gene chip detection kit was determined. Results: The effective hybridization signals were observed in the metabolism genes of two kinds of drugs detected by gene chips;the minimum detection concentration of PCR amplification products was 10³ copies·mL^-1 detected by gene chip;the coincidence rate of simultaneous determination of three chips in different batches and parallel measurement of 10 chips in the same batch was 100%;the chip was stable and could be stored for at least 6 months;the effective rate of gene chip detection kit in the 150 TD subjects from three regions was more than 90%. Conclusion: The high-throughput gene chip detection kit of individualized treatment of TD to detect the drug metabolism genes of clopidogrel and warfarin are successfully established,and the method has high sensitivity,satisfactory repeatability,strong stability and excellent preparation effect.

Objective: To observe the effects of sesamin on the abilities of learning and memory in the mice with Alzheimer's disease (AD), and to elucidate their possible mechanisms. Methods: Forty male C57BL/6 mice were randomly divided into control, model, piracetam, low dose of sesamin, and high dose of sesamin groups, and there were 8 mice in each group. The AD models were established by intracerebroventricular injection of amyloid β-protein fragment 25-35 (Aβ_25-35) in the mice in the other groups except control group. 24 h later, the mice in piracetam group were intragastrically administed with piracetam; the mice in low and high doses of sesamin groups were intragastrically administrated of 50 and 100 mg·kg^-1 sesamin, respectively; the mice in control and model groups were intragastrically administrated with the same amount of normal saline once a day for 2 weeks. Morris water maze test was used to observe the escape latencies,the swimming distance and the resident time in target quadrant of the mice in various groups;flow cytometry was used to detect the level of active oxygen(ROS) in brain tissue of the mice in various groups;ELISA was used to detect the activities of superoxide dismutase (SOD) and levels of malondialdehyde (MDA) in brain tissue of the mice in various groups; Western blotting was used to determine the expression levels of apoptosis-related protein B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cysteine aspartate specific proteinase-3 (Caspase-3) proteins in brain tissue of the mice in various groups. Results: Compared with control group,the escape latency of the mice in model group was significantly prolonged (P<0.05), and the swimming distance and the resident time in target quadrant were significantly shortened (P<0.05); compared with model group,the escape latencies of the mice in piracetam group, low dose of sesamin group and high dose of sesamin group were significantly shortened (P<0.05), and the swimming distance and the resident time in target quadrant were significantly increased (P<0.05). Compared with control group, the levels of ROS and MDA in brain tissue of the mice in model group were significantly increased (P<0.05), the SOD activity was significantly decreased (P<0.05), the expression levels of Bax and Caspase-3 protein were significantly increased (P<0.05), and the expression level of Bcl-2 protein and the value of Bcl-2/Bax were significantly decreased (P<0.05); compared with model group, the levels of ROS and MDA in brain tissues of the mice in piracetam, low dose of sesamin, and high dose of sesamin groups were significantly decresed (P<0.05), the activities of SOD were significantly increased (P<0.05), the expression levels of Bax and Caspase-3 proteins were significantly decresed (P<0.05), and the expression levels of Bcl-2 protein and the values of Bcl-2/Bax were significantly increased (P<0.05). Conclusion: Sesamin may improve the learning and memory abilities of the AD model mice by increasing the neuronal antioxidant capacity and inhibiting the neuronal apoptosis.

Objective: To investigate the inhibitory effect of SF2523, a dual inhibitor of BRD4 and PI3K, on the proliferation of human glioma stem cells TS576, and to preliminarily elucidate its mechanism. Methods: The human glioma stem cells TS576 were cultured and divided into control group and 0.25, 0.50,1.00 2.00 μmol·L^-1 SF2523 groups,and CCK-8 method was used to detect the survival rates of TS576 cells.The TS576 cells were divided into control group and 2 μmol·L^-1 SF2523 group,and the number of TS576 cells in various groups was detected by cell growth counting method.The TS576 cells were divided into control group, 1 and 2 μmol·L^-1 SF2523 groups,and the percentages of TS576 cells at different cell cycles in various groups were examined by flow cytometry.The TS576 cells were divided into control group, 1 and 2 μmol·L^-1 SF2523 groups,and the apoptotic rates of TS576 cells in various groups were detected by Annexin Ⅴ/PI staining. The TS576 cells were divided into control group, 1 and 2 μmol·L^-1 SF2523 groups, and the expression levels of cyclinD1, B-cell lymphoma-2(Bcl-2) and Bcl-2 associated X protein (Bax)proteins in the TS576 cells in various groups were detected by Western blotting method. Results: Compared with control group, the proliferation rates of TS576 cells in 0.25, 0.50,1.00 and 2.00 μmol·L^-1 SF2523 groups at 24, 48 and 72 h after treatment were significantly decreased (P<0.01).Compared with control group, the number of TS576 cells in 2 μmol·L^-1 SF2523 group was decreased significantly (P<0.05 or P<0.01). Compared with control group, the percentages of TS576 cells at G₁ phase in 1 and 2 μmol·L^-1 SF2523 groups were increased (P<0.05), the percentage of TS576 cells at S phase was decreased (P<0.05).The results of Annexin Ⅴ/PI staining showed that the apoptotic rates of TS576 cells in 1 and 2 μmol·L^-1 SF2523 groups were significantly increased at 72 h after treatment compared with control group (P<0.01). The results of Western blotting method showed that the expression levels of Bax protein in the TS576 cells in 1 and 2 μmol·L^-1 SF2523 groups were significantly increased (P<0.01), the expression levels of Bcl-2 and cyclinD1 were significantly decreased (P<0.05 or P<0.01), and the ratio of Bax/bcl-2 was also significantly increased (P<0.01). Conclusion: SF2523 can induce the G₁ phase arrest of TS576 cells by down-regulating the expression of cyclinD1 and promote the apoptosis of TS576 cells by up-regulating the expression of Bax and down-regulating the expression of Bcl-2, and then inhibit the proliferation of TS576 cells.

Objective: To investigate the effect of CRISPR/Cas9-mediated tyrosine kinase inhibitor(TKI) on the sensitivity of non-small cell lung cancer gene targeting gefifinib resistance, and to clarify its mechanism. Methods: The A549 cell strains knocked out tyrosine kinases(TKs) by using CRISPR/Cas9 system were established, and then divided into the A549, A549TKs^-/+ and A549 TKs^-/- cell strains according to the expression levels of TKI. The expression levels of P53, MDM2, Bcl-2 and Bax proteins in cell starins were detected by Western blotting method,the activities of A549, A549TKs^-/+, and A549 TKs^-/- cells were detected by CCK-8 method,the cell migration of the A549, A549TKs^-/+, and A549 TKs^-/- cells were detected by cell scratch method, the apoptotic rates of the A549, A549TKs^-/+, and A549 TKs^-/- cells were detected by flow cytometry,and the colony formation status of the A549, A549TKs^-/+, and A549^-/- cells were detected by colony formation assay. Results: The TKs knockout A549 cell strains were successfully established by CRISPR/Cas9 system. There were no statistically significant differences in the activites of the A549, A549TKs^-/+ and A549TKs^-/- cells (P>0.05),there were no significant differences in the apoptostic rates of the A549, A549TKs^-/+ and A549 TKs^-/- cells (P>0.05),and there was no statistically significant difference in the migration of the A549, A549TKs^-/+ and A549TKs^-/- cells. After intervented with gefitinib for 72 h,compared with A549 cell strain, the cell activities of the A549TKs^-/+ and A549TKs^-/- cell strains were significantly decreased(P<0.05),the apoptotic rates were significantly increased (P<0.05), the rates of scratch healing were obviously decreased(P<0.05),and the migration abilities were significantly decreased;compared with A549 cell stain,the expression levels of P53 and Bax proteins in the A549TKs^-/+ and A549TKs^-/- cell strains were significantly decreased (P<0.05),the expression levels of MDM2 and Bcl-2 proteins were significantly increased (P<0.05), and the colony formation rates were significantly decreased (P<0.05). Conclusion: Gefitinib intervention in the knockout A549 cell strain can decrease the cell viability and migration ability and increase the apoptotic rate and sensitivity of gefitinib resistance.

Objective: To investigate the effects of Dickkopf1(DKK1) protein on the biological behaviors of SBC-3 cells of human small cell lung cancer(SCLC),and to elucidate their mechanisms. Methods: The human SCLC cell strain SBC-3 were infected with lentiviruses over-expressing DKK1 and control viruses,respectively,and the cells stably expressing DKK1 protein (SBC-3-DKK1 group) and control cells (SBC-3-NC group) were obtained.RT-PCR and Western blotting methods were used to detect the expression levels of DKK1 mRNA and the expression amount of DKK1 protein in the SBC-3 cells in two groups;plate cloning assay was used to detect the colony formation rate of the cells in two groups;Transwell assay was used to observe the abilities of migration and invasion of the SBC-3 cells in two groups;Western blotting method was used to detect the expression amounts of MMP-9 protein in the SBC-3 cells in two groups. Results: The green fluorescence rate in the SBC-3 cells after lentivirus infection for 72 h was more than 80%.Compared with SBC-3-NC group,the expression level of DKK1 mRNA (P=0.004)and the expression amount of DKK1 protein in the SBC-3 cells in SBC-3-DKK1 group were significantly increased,the colony formation rate was significantly increased (P=0.002 6),the number of migration cells was significantly increased (P=0.006 2),the number of invadsion cells was significantly increased (P=0.021 4),and the expression amount of MMP-9 protein was significantly increased. Conclusion: Over-expression of DKK1 protein can promote the proliferation,migration and invasion of SBC-3 cells,and their mechanisms may be related to the up-regulation of MMP-9 protein expression.

Objective: To observe the expressions and distribution of Otos in cochlea tissue of the mice with age-related hearing loss (AHL) at different ages,and to explore its relationship with AHL. Methods: A total of 40 SPF C57BL/6Cnc mice were divided into 4-week-old group,16-week-old group,32-week-old group and 48-week-old group,and there were 10 mice in each group. The auditory brainstem response (ABR) was performed in the mice in various groups,then after the cochlea was removed,the expressions and distribution of Otospiralin protein in cochlea tissue of the mice in various groups were detected by immunofluorescence;the expression levels of Otos mRNA in cochlea tissue of the mice in various groups were examined by qRT-PCR method,and the expression levels of Otospiralin protein in cochlea tissue of the mice in various groups were detected by Western blotting method. Results: The Otospiralin proein was mainly expressed in the spiral ligament of the cochlea tissue of the mice and was expressed in all five fibrocytes;compared with 4-week-old group,the expression amounts of Otospiralin protein in cochlea tissue of the mice in 16-,32-,and 48-week-old groups were significantly increased(P<0.01).Compared with 4-week-old group,the mean ABR thresholds of the mice in 16-,32- and 48-week-old groups were significantly increased (P<0.01),and the expression levels of Otos mRNA and Otospiralin protein in cochlea tissue of the mice were significantly decreased (P<0.01);compared with 16-week-old group,the mean ABR thresholds of the mice in 32- and 48-week-old groups were significantly increased (P<0.01),and the expression levels of Otos mRNA and Otospiralin protein in cochlea tissue of the mice were significantly decreased (P<0.01); compared with 32-week-old group,the mean ABR threshold of the mice in 48-week-old group was significantly increased (P<0.01),and the expression levels of Otos mRNA and Otospiralin protein in cochlea tissue of the mice were significantly decreased (P<0.01). Conclusion: With the increasing of age,the auditory function of AHL mice is declined gradually,and the expression levels of Otos mRNA and Otospiralin protein are decreased gradually.The changes of Otos may be involved in the occurrence and development of AHL.

Objective: To observe the anti-tumor effect of different doses of ¹²⁵I seed implantation in the human breast cancer bearing nude mice, and to explore the mechanism. Methods: The healthy BALB/c nude mice were selected,and the human breast cancer HMLER90hi cells were transplanted into the fourth breast fat pad to establish the tumor-bearing models of nude mice. After successful modeling, 40 nude mice were randomly divided into:blank control group and low, middle and high doses of ¹²⁵I particle groups,and there were 10 rats in each group. The mice in blank control group were only implanted with one blank particle (without radioactive elements); the mice in low, middle and high doses of ¹²⁵I particle groups were implanted with 1.48×10^-7, 2.22×10^-7 and 2.96×10^-7 Bq ¹²⁵I particles, respectively, according to the Paris system principle. The volumes and weights of tumor of the mice in various groups were detected,and the inhibitory rates of growth of tumor of the mice in various groups were calculated.The levels of telomerase protein in tumor tissue of the mice in various groups were determined by ELISA method,the expression levels of CD90 and granulocyte-macrophage colony stimulating factor (GM-CSF)mRNAin the HMLER90hi cells of the mice in various groups were detected by qRT-PCR method. Results: At 7, 14 and 28 d after implantation of ¹²⁵I particles, the weights and volumes of tumor of the mice in different doses of ¹²⁵I particle groups were decreased compared with blank control group(P<0.05 or P<0.01), and the inhibitory rates of growth of tumor of the mice were significantly increased (P<0.05 or P<0.01);the ELISA results showed that compared with blank control group, the levels of telomerase protein in tumor tissue of the mice in different doses of ¹²⁵I particle groups were significantly decreased(P<0.05 or P<0.01);the qRT-PCR results showed that compared with blank control group, the expression levels of CD90 and GM-CSF mRNA in the HMLER90hi cells of the mice in different doses of ¹²⁵I particle groups were significantly decreased(P<0.05 or P<0.01). Conclusion: Different doses of ¹²⁵I particles can inhibit the proliferation of breast cancer in the nude mice, and its mechanism may be related to the inhibition of expressions of CD90 and GM-CSF mRNA in tumor-bearing tissue.

Objective: To investigate the protective effect of soy isoflavone on the oxidative stress injury of the perimenopause model rats with type 2 diabetes mellitus, and to elucidate its mechanism. Methods: Ten rats of all the sixty rats were randomly choosen as control group(n=10).The perimenopause models of rats with type 2 diabetes mellitus were established by feeding high sugar diet and removing both ovaries,and the rest 46 perimenopause model rats were randomly divided into sham operation group(n=9), model group(n=8), low(n=9), medium (n=10)and high(n=10) doses of soy isoflavone groups. The partial adipose tissue below both ovaries of the rats in sham operation group was removed, the rats in low, medium and high doses of soy isoflavone groups were given different doses (90, 180, and 360 mg·kg^-1) of soy isoflavone by gavage, while the rats in control group, sham operation group and model group were given the same volume of normal saline, lasted for 8 weeks. The body weights and the levels of fasting blood glucose (FBG) of the rats in various groups were measured.The levels of serum estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), nitric oxide (NO), malondialdehyde (MDA), and reactive oxygen species (ROS) of the rats in various groups were measured by ELISA method. Results: Compared with model group, the body weights and the FBG levels of the rats in low, medium and high doses of soy isoflavone groups were increased significantly (P<0.05); the levels of serum E2 were significantly increased (P<0.05),the levels of serum FSH and LH were significantly decreased (P<0.05), the levels of serum SOD, GSH-Px and NO were significantly increased (P<0.05), and the levels of serum MDA and ROS were significantly decreased (P<0.05). Conclusion: Soy isoflavone can regulate the level of serum estrogen, improve the ability of anti-oxidative stress, and protect the oxidative stress injury in the perimenopause model rats with type 2 diabetes mellitus in a non-dose-dependent manner.

Objective: To investigate the expressions of RhoA and ROCK proteins in glomeruli of the C57BL/6J young mice fed with high-fat diet(HFD),and to elucidate whether the RhoA/ROCK pathwaywas related to glomerular injuryinduced by HFD. Methods: Thirty-six male C57BL/6J mice were randomly divided into normal control group and HFD group,and there were 18 mice in each group.The mice in normal control group were given standard diet,and the mice in HFD group were given HFD;they were fed for 12 weeks.The body weights and the serum levels of total cholesterol (TC) and triglyceride (TG) of the young mice in two groups were measured.HE staining,PAS staining and Masson staining were used to observe the pathomorphology of kidney tissue,the relative areas of mesangial membrane and basement membrane and renal interstitial fibrous tissue of the young mice in two groups were detected;immunohistochemical method was used to detect the expression levels of RhoA and ROCK in glomeruli of the young mice in two groups.The expression levels of RhoA,ROCK and vimentin proteins in glomeruli of the young mice in two groups were detected by Western blotting method. Results: Compared with normal control group,the body weight,the serum TC and TG levels of the young mice in HFD group were significantly increased (P<0.01).The HE staining results showed obvious fatty degeneration in the glomeruli of the young mice in HFD group;the PAS staining results showed mild proliferation of glomerular mesangial matrix of the young mice in HFD group;the Masson staining results showed that blue collagen fibers in glomeruli of the mice in HFD group were more abundant than those of the mice in normal control group.Compared with normal control group,the relative area of mesangial membrane and basement membrane of the young mice in HFD group was significantly increased (P<0.05),and the relative area of renal interstitial fibrous tissue was increased (P<0.05).Compared with normal control group,the expression levels of RhoA and ROCK proteins in the glomeruli of the young mice in HFD group were increased (P<0.05);the expression levels of RhoA,ROCK and Vimentin in the glomeruli of the young mice in HFD group were increased (P<0.05). Conclusion: The high expressions of RhoA and ROCK proteins in glomeruli of the C57BL/6J mice fed with HFD may be one of the causes of glomerular injury.

Objective: To investigate the role of long non-coding RNA(lncRNA) MALAT1 in the proliferation and migration of YAP-mediated non-small cell lung cancer (NSCLC) cells,and to elucidate its mechanism. Methods: The A549 and H1299 cell lines were selected and divided into Scramble siRNA group(transfected with Scramble siRNA),siYAP-1 group(transfected with siYAP-1)and siYAP-2 group(transfected with siYAP-2). RT-PCR and Western blotting methods were used to detect the expression levels of YAP and MALAT1 mRNA and YAP protein in the cells in variouis groups. The pcDNA3.1-YAP plasmid(pcDNA3.1-YAP group), pcDNA3.1-YAP plasmid plus siMALAT1-2 (pcDNA3.1-YAP+siMALAT1-2 group)and the control plasmid (pcDNA3.1-YAP+Scramble siRNA group)were transfected into the A549 and H1299 cells, respectively.CCK-8 method and scratch assay were used to detect the proliferation and migration abilities of the cells in various groups. Results: The expression levels of YAP mRNA and protein in the A549 and H1299 cells in siYAP-1 and siYAP-2 groups were significantly lower than those in Scramble siRNA group(P<0.05), and the expression levels of MALAT1 mRNA in the cells in siYAP-1 and siYAP-2 groups were significantly lower than those in Scramble siRNA group (P<0.05).Compared with pcDNA3.1-YAP group, the proliferation abilities of the A549 and H1299 cells in pcDNA3.1-YAP+siMALAT1-2 group were decreased(P<0.05),the migration ability of the H1299 cells in pcDNA3.1-YAP+siMALAT1-2 group was decreased(P<0.05). Conclusion: In the NSCLC cells, YAP promotes the cell proliferation and migration by regulating the expression of lncRNA MALAT1.

Objective: To observe the developmental situation of the lung of the neonatal rats with chronic bronchopulmonary dysplasia (BPD) after treated with Radix Salviae Miltiorrhizae Decoction,and to clarify the effect of Radix Salviae Miltiorrhizae Decoction on the BPD in the neonatal rats induced by high-volume fractinal oxygen(hyperoxia) and its mechanism. Methods: A total of 200 neonatal rats were divided into control group,model group,dexamethasone group,low and high doses of Radix Salviae Miltiorrhizae Decoction groups,and there were 40 rats in each group.Except for the rats in control group,the rats in the other four groups were induced by continuous inhalation of hyperoxia.The rats in dexamethasone group and Radix Salviae Miltiorrhizae Decoction group were treated with dexamethasone solutions (0.75 μg·kg^-1) and 6.00 amd 24.00 mg·kg^-1 Radix Salviae Miltiorrhizae Decoction,respectively,and the rats in control group were given the equal volume of normal saline.After 21 d of administration,the wet mass/dry mass (W/D) values of lung tissue of the rats in various groups were measured,the pathomorphology of lung tissue of the rats in various groups were observed by HE staining,and the number of radical alveolar counts (RAC) in lung tissue of the rats in various groups was calculated;the levels of malondialdehyde (MDA),superoxidedismutase (SOD) and glutathione peroxidase (GSH-Px) in lung tissue of the rats in various groups were detected,the levels of interleukin-1 (IL-1),interleukin-10(IL-10) and tumor necrosis factor-α(TNF-α) in lung tissue of the rats in various groups were determined by ELISA method,the expression levels of Bax,Bcl-2 and caspase-3 proteins in lung tissue of the rats in various groups were measured by Western blotting method. Results: Compared with control group,the W/D value of lung tissue of the rats in model group was increased significantly (t=3.144,P<0.05),and the obvious pulmonary edema was found.After 21 d of oxygen exposure,the lung tissue of the rats in model group was shown as enlarged alveolar cavity, simplified structure,and thickened interval,and the number of RAC was decreased significantly(t=3.989,P<0.05);the inflammatory cell infiltration and the levels of MDA,SOD,GSH-Px,IL-1β,and TNF-α and the expression levels of Bax and caspase-3 proteins in lung tissue of the rats in model group were increased significantly (t=6.562,t=1.971,t=1.972,t=20.241,t=32.808,t=22.663,t=19.234,P<0.05),and the level of IL-10 in lung tissue of the rats was decreased significantly (t=7.857,P<0.05).Compared with model group,the degrees of pulmonary edema of the rats in dexamethasone and Radix Salviae Miltiorrhizae Decoction groups were improved,and the W/D values of lung tissue were significantly decreased(t=1.018,t=2.691,t=0.760,P<0.05),and the alveolar structural disorder was alleviated; the levels of MDA,SOD,GSH-Px,IL-1,and TNF-α and the expression levels of Bax and caspase-3 proteins in lung tissue of the rats were decreased (t=6.562,t=1.971,t=1.972,t=20.241,t=32.808,t=22.663,t=19.234,P<0.05),the levels of IL-10 and the expression levels of Bcl-2 protein were increased(t=7.857,t=7.743,P<0.05),expecially in high dose of Radix Salviae Miltiorrhizae Decoction group. Conclusion: Radix Salviae Miltiorrhizae Decoction can effectively protect the bronchus and lung injuries induced by hyperoxia in the neonatal rats.

Objective: To investigate the effect of serum melatonin level in the rats with pulpitis on the nucleotide-binding oligomerization domain-like receptor protein 3/cysteine-containing aspartate proteolytic enzyme 1 (NLRP3/caspase-1) signaling pathway,and to explore the mechanism of the effect of melatonin on pulpitis. Methods: A total of 130 rats were divided into control group (n=40),pulpitis group (n=60) and pulpitis+melatonin group (n=30).Thirty rats with pulpitis were randomly divided into pulpitis 1 d group,pulpitis 3 d group and pulpitis 5 d group.The pulpitis rat model was established by pulpotomy exposure method, and the rats in pulpitis+melatonin group were intraperitoneally injected with melatonin after intramedullary surgery.HE staining was used to confirm the modeling of pulpitis;the serum levels of interleukin-1β(IL-1β) and melatonin and the levels of IL-1β in dental pulp tissue of the rats in various groups were detected by ELISA method, the expresion levels of NLRP3 and caspase-1 mRNA in dental pulp tissue of the rats in various groups were detected by RT-PCR method,and the expression levels of NLRP3 and caspase-1 proteins in dental pulp tissue of the rats in various groups were detected by Western blotting method. Results: The HE staining results showed that in control group,the dental pulp tissue of the rats was normal;in pulpitis group,the cavities in the dental pulp and a large number of inflammatory cells were found.The serum IL-1β levels of the rats in pulpitis 1 d group and pulpitis 3 d group were obviously higher than those in control group (P<0.05),and the serum melatonin levels were obviously lower than those in control group (P<0.05);the serum IL-1β level of the rats in pulpitis 3 d group was obviously lower than that in pulpitis 1 d group (P<0.05),and the serum melatonin level was obviously higher than that in pulpitis 1 d group (P<0.05).The levels of IL-1β in dental pulp tissue of the rats in pulpitis group and pulpitis+melatonin group were obviously higher than those in control group (P<0.05),the level of IL-1β in dental pulp tissue of the rats in pulpitis+melatonin group was obviously lower than that in pulpitis group (P<0.05). The expression levels of NLRP3 and caspase-1 mRNA and proteins in dental pulp tissue of the rats in pulpitis group and pulpitis+ melatonin group were obviously higher than those in control group (P<0.05),and the levels of NLRP3 and caspase-1 mRNA and proteins in dental pulp tissue of the rats in pulpitis+melatonin group were obviously lower than those in pulpitis group (P<0.05). Conclusion: The serum melatonin level in the rats with pulpitis is reduced,and melatonin can reduce the inflammatory response in dental pulp tissue of the rats with pulpitis by inhibiting NLRP3/caspase-1 signaling pathway.

Objective: To investigate the effect of daunorubicin on the apoptosis of human B-cell lymphoma OCI-LY7 cells and its mechanism,and to elucidate the drug resistance mechanism of daunorubicin in the diffuse large B-cell lymphoma. Methods: The OCI-LY7 cells cultured in vitro were divided into 0,0.1,1.0 and 10.0 mg·L^-1 daunorubicin groups.The survival rates of OCI-LY7 cells in various groups were detected by MTT assay,the apoptotic rates of the OCI-LY7 cells in various groups were detected by flow cytometry,and the expression levels of apoptosis-related proteins Bax,cleaved caspase-3, and survivin in the OCI-LY7 cells in various groups were detected by Western blotting method. The follow-up experiments were divided into control group,daunorubicin group,daunorubicin+vector group and daunorubicin+survivin group; the survival rates,the apoptotic rates and the expression levels of apoptosis-related proteins in the OCI-LY7 cells in various groups after overexpression of survivin were detected by the above-mentioned methods. Results: Compared with 0 mg·L^-1 group,the survival rates of OCI-LY7 cells and the expression levels of survivin protein in the OCI-LY7 cells in 0.1,1.0 and 10.0 mg·L^-1 daunorubicin groups were decreased gradually after treated for 48 h(P<0.05),while the apoptotic rates and the expression levels of Cleaved caspase-3 and Bax proteins in the OCI-LY7 cells were gradually increased (P<0.05). Compared with control group,the survival rate and the expression level of survivin protein in the OCI-LY7 cells in daunorubicin group were significantly decreased(P<0.05),while the apoptotic rate and the expression levels of cleaved caspase-3 and Bax proteins in the OCI-LY7 cells were significantly increased (P<0.05); compared with daunorubicin group,the survival rate and the expression level of survivin protein in the OCI-LY7 cells in daunorubicin+survivin group were significantly increased(P<0.05),while the apoptotic rate and the expression levels of cleaved caspase-3 and Bax protein in the OCI-LY7 cells in daunorubicin+vector group were significantly decreased (P<0.05). Conclusion: Daunorubicin can induce the apoptosis of OCI-LY7 cells,and its mechanism may be related to down-regulation of the expression of survivin protein.

Objective: To investigate the effect of miR-302b-3p on the proliferation and apoptosis of the esophageal cancer EC-109 cells,and to elucidate the related mechanism of miR-302b-3p inhibiting the proliferation and inducing apoptosis of the EC-109 cells. Methods: The esophageal cancer EC-109 cells and normal esophageal epithelium HET-1A cells were cultured in vitro.The expression levels of miR-302b-3p in the EC-109 cells and HET-1A cells were detected by real-time fluorescence quantitative PCR method.The EC-109 cells were transfected instantaneously and divided into blank control group(non-transfection),miR-NC group (transfected with negative control of miR-302b-3p mimics),miR-302b-3p group (transfected with miR-302b-3p mimics),anti-miR-NC group (transfected with negative control of miR-302b-3p inhibitor) and anti-miR-302b-3p group (transfected with miR-302b-3p inhibitor).The viabilities of the EC-109 cells in various groups were detected by MTT assay;the percentages of EC-109 cells at different cell cycles and the apoptotic rates of EC-109 cells were detected by flow cytometry;the expression levels of CyclinD1,CDK2,Bax,and Cleaved caspase-3 in the EC-109 cells in various groups were detected by Western blotting method;the luciferase activities of EC-109 cells in various groups were detected by double luciferase reporter gene assay and Western blotting method was used to detect the expression level of epithelial cell transformation sequence 2(ECT2) protein in the EC-109 cells in various groups. Results: Compared with the HET-1A cells,the expression level of miR-302b-3p in the EC-109 cells was significantly decreased (P<0.05). Compared with blank control group,the viability of EC-109 cells,the percentage of cells at S phase and the expression levels of CyclinD1 and CDK2 protein in the EC-109 cells in miR-302b-3p group were significantly decreased(P<0.05),while the percentage of the cells at G₀/G₁ phase,the apoptotic rate,and the expression levels of Bax and Cleaved caspase-3 proteins in the EC-109 cells were significantly increased(P<0.05).Compared with anti-miR-NC group, the cell viability,the percentage of the cells at S phase and the expression levels of Cyclin D1 and CDK2 protein in the EC-109 cells in anti-miR-302b-3p group were significantly increased(P<0.05),and the percentage of the cells at G₀/G₁ phase,the apoptotic rate and the expression levels of Bax and Cleaved caspase-3 proteins in the EC-109 cells were significantly decreased (P<0.05).Compared with miR-NC+ECT2-WT group,the luciferase activity of the EC-109 cells in miR-302b-3p+ECT2-WT group was decreased(P<0.05);compared with anti-miR-NC+ECT2-WT group,the luciferase activity of the EC-109 cells in anti-miR-302b-3p+ECT2-WT group was increased(P<0.05). Conclusion: miR-302b-3p can inhibit the proliferation and induce the apoptosis of EC-109 cells,and its mechanism may be related to the targeting regulation of ECT2 protein expression.

Objective: To investigate the effects of miR-367 on the proliferation, invasion and migration of the human breast cancer MCF-7 cells, and to elucidate their mechanisms. Methods: The relative expression levels of miR-367 in the breast cancer tissues, adjacent tissues, MCF-7 cells and MCF-10A cells were detected. The MCF-7 cells were transfected with miR-NC or miR-367-inhibitor as negative control group and miR-367-inhibitor group, and the cell viabilities, the number of colonies, the positive expression rates of Ki67, the rate of cell wound healing, the number of invasive cells and the relaitive expression levels of KLF4 in the MCF-7 cells in two groups were detected. The MCF-7 cells were transfected with miR-NC or miR-367-mimic as negative control group and miR-367-mimic group, and the number of colonies, the rates of cell wound healing, the number of invasive cells and the relaitive expression levels of KLF4 in the MCF-7 cells in two groups were detected. Results: Compared with adjacent tissue or MCF-10A cells, the relative expression levels of miR-367 in breast cancer tissues and MCF-7 cells were significantly increased (P<0.01).Compared with negative control group,the relative expression level of miR-367 in the MCF-7 cells, the cell viability,the number of colonies,the positive expression rate of Ki67, the rate of cell wound healing and the number of invasive cells in miR-367-inhibitor group were significantly decreased (P<0.01), and the relative expression level of KLF4 was significantly increased (P<0.01);compared with positive control group,the relative expression level of miR-367 in the MCF-7 cells, the number of colonies, rates of cell wound healing and number of invasive cells in miR-367-mimic group were significantly increased (P<0.01), and the relative expression level of KLF4 was significantly decreased (P<0.01). Conclusion: miR-367 promotes the proliferation, migration and invasion of MCF-7 cells, and the mechanism may be related to the inhibition of KLF4 expression.

Objective: To observe the expressions of heparanase(HPA) protein and integrin α5β1 protein in the human benign and malignant hydatidform mole tissues,and to elucidate their roles in the occurrence and development of benign and malignant hydatidiform moles. Methods: A total of 50 cases of normal placental villus tissue (normal placental villus group),40 cases of benign hydatidiform moles tissue (benign hydatidform mole group) and 17 cases of malignant hydatidiform mole tissue (malignant hydatidform mole group) were selected. The positive expression rates and expression intensities of HPA protein and integrin α5β1 protein in tissues in various groups were detected by immunohistochemical SP method, the expression levels of HPA protein and integrin α5β1 proteins in tissues in various groups were detected by Western blotting method; Spearman correlation method was used to analyze the correlation between the expressions of HPA protein and integrin α5β1 protein. Results: The positive expression rates,the expression intensities and the expression levels of HPA and integrinα5β1 in tissues in benign hydatidiform mole group and malignant hydatidiform mole group were higher than those in normal placental villus group(P<0.05); the positive expression rate,the expression intensity, and the expression levels of HPA protein and integrin α5β1 protein in tissue in malignant hydatidiform mole group were higher than those in benign hydatidiform mole group(P<0.05).There were positive correlations between the expressions of HPA protein and integrin α5β1 protein in both benign and malignant hydatidiform mole tissues(r=0.506,P<0.05;r=0.555,P<0.05). Conclusion: The combined detection of the expressions of HPA protein and integrin α5β1 protein may become a predictive indicator for the differential diagnosis of benign hydatidiform mole and malignant hydatidiform mole.

Objective: To discuss the effects of febuxostat on the serum uric acid level and the expression level of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) protein in the hyperuricemia model rats, and to elucidate their mechanisms. Methods: A total of 45 rats were randomly divided into control group, hyperuricemia group and febuxostat group,and there were 15 rats in each group. The rat models of hyperuricemia were established in hyperuricemia group and febuxostat group by intragastric administration of potassium oxonate. The rats in febuxostat group were given a daily dose of 7.2 mg·kg^-1 febuxostat,and the rats in control group were administrated with the same volume of normal saline,lasted for 4 weeks. HE staining was used to observe the pathomorphology of kidney tissue of the rats in various groups, automatic biochemical analyzer was used to detect the levels of serum uric acid, creatinine and urea nitrogen of the rats in various groups,ELISA method was used to determine the levels of serum interleukin-1β (IL-1β) and interleukin-18(IL-18) of the rats in various groups,and immunohistochemistry method was performed to detect the expression levels of apoptosis-associated speck-like protein (ASC) and NLRP3 proteins in kidney tissue of the rats in various groups. Results: Compared with control group, the urine volume and water intake of the rats in hyperuricemia group were increased (t=5.317,t=3.674,P<0.05), the body weight was decreased (t=6.374,P<0.05), the levels of serum uric acid, creatinine and urea nitrogen were increased (t=21.463,t=15.342,t=4.603,P<0.05),the serum IL-1β and IL-18 levels were increased (t=35.761,t=44.168,P<0.05),and the expression levels of ASC and NLRP3 protein in kidney tissue were increased (t=17.064,t=26.314,P<0.05);compared with hyperuricemia group, the urine volume and water intake of the rats in febuxostat group were decreased (t=4.027,t=2.976,P<0.05),the body weight was increased (t=3.694,P<0.05),the serum blood uric acid, creatinine and urea nitrogen levels were decreased (t=13.064,t=7.461,t=3.528,P<0.05),the serum IL-1β and IL-18 levels were decreased (t=24.162,t=17.314,P<0.05),and the expression levels of ASC and NLRP3 protein in kidney tissue were decreased (t=8.061,t=11.057,P<0.05). Conclusion: Febuxostat may play a kidney protective role in the hyperuricemia model rats by inhibiting the activation of NLRP3 inflammatory bodies and the levels of its downstream inflammatory factors.

Objective: To study the effects of curcumin on the cognitive function and Toll-like receptor 4/nuclear transcription factor-κB (TLR4/NF-κB) pathway in hippocampus tissue of the mice with sleep deprivation, and to explore the mechanism of curcumin in the treatment of sleep deprivation. Methods: A total of 100 mice were randomly divided into control group, model group and low,middle, high doses of curcumin groups,and there were 20 mice in each group.Except control group,the mice in the other groups were established sleep deprivation models by modified multi-platform water environment sleep deprivation method;after modeling, the mice in low, meddle and high doses of curcumin groups were intraperitoneally injected with 5, 10, and 20 mg·kg^-1 curcumin, once a day for 3 d. Morris water maze test was used to determine the learning and memory abilities of the mice in various groups,and open-field test was used to determine the rearing and crossing scores of the mice in various groups; the levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the hippocampus tissue of the mice in various groups were detected by ELISA method, the expression levels of TLR4 and NF-κB p65 proteins in hippocampus tissue of the mice in various groups were detected by Western blotting method, and the expression levels of IL-1β, IL-6, TLR4, and NF-κB mRNA in hippocampus tissue of the mice in various groups were detected by RT-PCR method. Results: Compared with model group, the escape latencies of the mice in different doses of curcumin groups were shortened (P<0.05), the time of staying on the original platform quadrant was prolonged(P<0.05), the times of crossing the original platform were increased (P<0.05), the rearing and crossing scores of the mice in open-field test were decreased (P<0.05),the expression levels of IL-1β, IL-6, TLR4 and NF-κB p56 proteins in hippocampus tissue of the mice in various groups were significantly decreased (P<0.05),and the expression levels of IL-1β, IL-6, TLR4 and NF-κB mRNA in hippocampus tissue of the mice in various groups were decreased(P<0.05). Conclusion: Curcumin may improve the cognitive function in the mice with sleep deprivation by inhibiting the TLR4/NF-κB pathway.

Objective: To investigate the effect of inhibiting TRIM24 gene expression on the proliferation, apoptosis, invasion and migration of breast cancer MCF-7 cells, and to elucidate the role of TRIM24 gene in the occurrence and development of breast cancer. Methods: The TRIM24 siRNA and negative control NC-siRNA were transfected into the MCF-7 cells by liposome method, and used as si-TRIM24 group and NC-siRNA group, respectively. The untransfected MCF-7 cells were used as control group. qRT-PCR and Western blotting methods were used to detect the expression levels of TRIM24 mRNA and protein in the MCF-7 cells in various groups after transfected for 48 h,MTT assay was performed to detect the proliferation activities of the MCF-7 cells in various groups,flow cytometry was used to determine the apoptotic rates of the MCF-7 cells in various groups,and Transwell assay was used to measure the number of invasion and migration cells in various groups. Results: Compared with control group and NC-siRNA group, the expression levels of and protein in the MCF-7 cells were significantly decreased (P<0.05), and the proliferation activity of the MCF-7 cells was decreased (P<0.05), the apoptotic rate of the MCF-7 cells was significantly increased (P<0.05), and the number of invasion and migration MCF-7 cells was significantly decreased (P<0.05). Conclusion: Inhibition of the expression of TRIM24 gene can significantly inhibit the proliferation, invasion and migration of breast cancer MCF-7 cells, and induce the apoptosis.

Objective: To observe the effect of Compound Sophora Flavescens Injection on the radiosensitivity of the human nasopharyngeal carcinoma CNE-2 cells,and to explore its mechanism. Methods: The human nasopharyngeal carcinoma CNE-2 cells were cultured in vitro. The inhibitory rates of proliferation of CNE-2 cells after treated with different concentrations of Compound Sophora Flavescens Injection for 24, 48, and 72 h were detected by MTT method. The semi-inhibitory concentration (IC₅₀) of Compound Sophora Flavescens Injection on CNE-2 cells was calculated. The CNE-2 cells were divided into control group(without irradiation and adminnistration), irradiation group (treated with 2 and 4 Gy X-ray irradiation) and combination group(treated with different concentrations of Compound Sophora Flavescens Injection and then received X-ray irradiation). MTT assay was used to detect the inhibitory rates of proliferation of the CNE-2 cells in various groups, flow cytometry was used to detect the apoptotic rates of the CNE-2 cells in various groups,and clone formation test was used to detect the survival rates of the CNE-2 cells in various groups. Results: After treated with different concentrations of Compound Kushen Injection for 24, 48 and 72 h, the inhibitory rates of proliferation of the CNE-2 cells were increased compared with the CNE-2 cells without Compound Kushen Injection treatment (P<0.05) in an dose-dependent manner;the inhibitory rate of proliferation of the CNE-2 cells after treated with Compound Kushen injection for 48 h was higher than 24 h (P<0.05).The apoptotic rate of the CNE-2 cells in radiation group was higher than that in control group (P<0.05); the apoptotic rate of the CNE-2 cells in combination group was higher than that in radiation group (P<0.05);the survival rate of the CNE-2 cells in combination group was lower than that in radiation group(P<0.05). Conclusion: Compound Sophora Flavescens Injection can promote the irradiation-induced apoptosis of nasopharyngeal carcinoma CNE-2 cells, therefore, Compound Sophora Flavescens Injection has the radiosensitization effect on the CNE-2 cells.

Objective: To observe the effects of body weight support treadmill training(BWSTT) with different exercise intensities on the expression levels of tyrosine protein kinase B (TrkB) and brains-derived neurotrophic factor (BDNF) proteins in spinal cord tissue and motor function of the spinal cord injury(SCI) rats, and to explore the possible mechanism. Methods: Fifty adult male SD rats were randomly divided into sham operation group, SCI group, BWSTT-A group(given low intersity exercise), BWSTT-B group (given middle intensity exercise)and BWSTT-C group (given high intersity exercise); there were ten rats in each group. the SCI model was prepared by using a self-made striker.The rats in sham operation group only underwent laminectomy to expose the dura mater and were sutured directly, and the rats didn't receive any treatment after surgery;the rats in SCI group weren't given any treatment after modeling;the rats in BWSTT groups were given rehabilitation exercise after SCI and the walking speeds of the rats in three groups were 7, 15, and 21 cm·s^-1, respectively.The recovery status of hind limb function of the rats in various groups was evaluated by Basso,Beattie,and Bresnahan(BBB) score 35 d after modeling;the expression levels of TrkB and BDNF proteins in spinal cord tissue of the rats in various groups were detected by Western blotting method,and the morphology and the number of Nissl bodies of the rats in various groups were analyzed by Nissl staining. Results: Compared with SCI group, the BBB scores of the rats in BWSTT-B and BWSTT-C groups were increased (P<0.01); compared with BWSTT-A group, the BBB scores of the rats in BWSTT-B and BWSTT-C groups were increased (P<0.01).Compared with sham operation group,the expression level of TrkB protein in spinal cord tissue of the rats in SCI group was decreased(P<0.01);compared with SCI group, the expression levels of TrkB protein in spinal cord tissue of the rats in BWSTT-B and BWSTT-C groups were increased (P<0.01). Compared with sham operation group, the expression level of BDNF protein in spinal cord tissue of the rats in SCI group were decreased(P<0.01); compared with SCI group, the expression levels of BDNF protein in spinal cord tissue of the rats in BWSTT-B group and BWSTT-C group were increased (P<0.01). The Nissl staining results showed that compared with SCI group, the number of Nissl cells in BWSTT groups was more and the morphology of cells was better. Conclusion: Middle and high intensities of BWSTT may promote the recovery of motor function of the SCI rats, and its mechanism may be related to up-regulation of the expressions of TrkB and BDNF proteins in spinal cord tissue.

Objective: To observe the effect of hypothalamus pituitary adrenal axis(HPA) disorder on the depression-like behavior and the expression of Caspase-3 protein in hippocampus tissue of the spinal cord injury(SCI) rats, and to explore its possible mechanism. Methods: A total of 40 male SD rats were rardomly divided into normal group (12 rats), sham model group (12 rats) and SCI group (16 rats). The SCI model was made by using self-made impactor. Open field test was used to detect the total distance of locomotor activities, sucrose preference test was used to detect the percentages of sucrose preference, forced swimming test was used to detect the immobility time of forced swimming, and ELISA method was used to detect the serum adrenocorticotropic hormone(ACTH) and corticosterone(CORT) levels of the rats in various groups. Results: The Open field test results showed that there was a significant difference in the total distances of locomotor activities of the rats between various groups at different time points (F=23.950, P=0.000); the sucrose preference of showed that there was a significant difference in the percentages of sucrose preference of the rats between various groups at different time points (F=11.99, P=0.001); compared with the baseline time point, the difference of percentages of sucrose preference of the rats in various groups on the 9th and 33rd days were increased(P<0.01);compared wtih sham model group,the percentages of sucrose preference of the rats in SCI group were significantly decreased(P<0.05).The forced swimming test results showed that the differences of immobility time of forced swimming of the rats in various groups had a statistically significant difference(F=104.281, P=0.000) at different time points, and there was a interaction between time and groups(F=58.036, P=0.000). The ELISA results showed that there was a significant difference in the serum CORT level of the rats between various groups at different time points(F=22.869, P=0.000), and there was a interaction between time and groups (F=7.753, P=0.000). There was a significant difference in the serum ACTH levels of the rats between various groups at different time points(F=36.598, P=0.000), and there was a interaction between time and groups (F=6.452, P=0.000). The expression levels of Caspase-3 protein in hippocampus tissue of the rats in various groups had a statistically significant difference (F=169.937, P=0.000); compared with normal group and sham model group, the level of Caspase-3 protein in hippocampus tissue of the rats in SCI group had a statistically significant difference (P<0.01). Conclusion: The hyperfunction of HPA axis in the SCI rats leads to the increase of Caspase-3 protein expression in hippocampus tissue, which leads to the depression-like behavior in the SCI rats, thus reducing the negative feedback regulation function of HPA axis and promoting the occurrence and development of depression of the SCI rats.

Objective: To analyze the dynamic changes of serum levels of macrophage inflammatory protein-1α (MIP-1α) and interleukin-13 (IL-13) of the patients with severe asthma, and to evaluate their clinical values in judgment of the short-term prognosis in the patients with severe asthma. Methods: A total of 102 patients with asthma were divided into severe asthma group (42 cases of severe asthma) and mild asthma group (60 cases of mild and moderate asthma)according to the degree of asthma; a total of 50 contemporaneous healthy examinees were selected as control group. After follow-up for one year, the patients in severe asthma group were divided into preservation sub-group and recurrence sub-group.The serum levels of MIP-1α, IL-13, interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) of the subjects in three groups were detected before therapy and on the 1st, 3rd,and 7th days after therapy respectively; the pulmonary indexes including forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), FEV1/FVC, maximum mid-expiratory flow (MMEF) and peak expiratory flow (PEF) of the subjects in three groups were also detected and compared. The correlations between the serum levels of MIP-1α and IL-13 on the 7th day after therapy and FEV1, MMEF and PEF were confirmed by Pearson linear correlation analysis. The serum levels of IL-13 and MIP-1α of the patients in severe ashma group on the 7th day after therapy were compared between preservation sub-group and recurrence sub-group, the relationships between the clinical indexes mentioned above and the recurrence rate of the patients after 1-year follow-up were confirmed by multivariate Logistic analysis,and the diagnostic values of the serum levels of MIP-1α and IL-13 in the recurrence of the patients with severe asthma were analyzed by receiver operating characteristic (ROC) curves. Results: With the prolongation of therapy time, the serum levels of -6, IL-13, TNF-α, and MIP-1α of the patients in severe asthma group and mild asthma group were decreased (P<0.05), and FEV1, FEV1/FVC, MMEF and PEF of the patients were increased (P<0.05). At the same time point, the serum levels of IL-6, IL-13, TNF-α, and MIP-1α of the patients in severe asthma group were higher than those in mild asthma group(P<0.05) and control group(P<0.05); FEV1, FEV1/FVC, MMEF, and PEF of the patients in severe asthma group were lower than those in mild asthma group(P<0.05) and control group (P<0.05). The Pearson linear correlation analysis results showed that there were negative correlations between serum MIP-1α, IL-13 and FEV1, MMEF, PEF (P<0.05). After 1-year follow-up, the serum levels of IL-13 and MIP-1α in the patients in preservation sub-group in severe asthma group were lower than those in recurrence sub-group (P<0.05). The multivariate Logistic analysis results showed that the differences of serum levels of MIP-1α and IL-13 were the independent risk factors of the recurrence of severe asthma (OR=2.135, P=0.033; OR=2.867, P=0.023). Conclusion: The decreasing degree of the serum levels of MIP-1α and IL-13 can assess the pulmonary function and the recurrence of the patients with severe asthma after 1-year follow-up.

Objective: To observe the effects of aspirin combined with electrical stimulation of pelvic floor smooth muscle on the endometrium of the intrauterine adhesion(IUA) patients after hysteroscopic adhesiolysis, and to clarify its improvement effect on the neovascularization and blood circulation after operation of the IUA patients. Methods: Eighty patients with moderate or severe IUA, having fertility intention, were divided into control group, drug group, electrical stimulation group and combination group(n=20), and all the patients underwent hysteroscopic adhesidysis. The patients in control group were treated with estrogen progesterone sequential therapy,the patients in drug group were treated with estrogen progesterone sequential therapy plus aspirin, the patients in electrical stimulation group were treated with estrogen progesterone sequential therapy combined with pelvic floor smooth muscle stimulation,and the patients in combination group were treated with estrogen progesterone sequential therapy plus aspirin combined with pelvic floor smooth muscle stimulation. The efficacies of patients in various groups before and after treatment were compared, and the endometrium thickness (ET), pulsatility index (PI), resistance index (RI), pregnancy status,and the endometrium microvessel density (MVD) values of the IUA patients in various groups were observed;the expression levels of vascular endothelial growth factor (VEGF) mRNA and protein in endometrial tissue of the IUA patients in various groups were detected by RT-qPCR and Western blotting methods. Results: The total effective rate of patients in combination group was significantly higher than those in control group, drug group and electricalal stimulation group(P<0.05). Compared with before treatment, the ET of the IUA patients in drug group, electrical stimulation group and combination group after treatment were increased significantly (P<0.05), and PI and RI were decreased significantly (P<0.05);after treatment,the PI and RI of the IUA patients in combination group were significantly lower than those in control group, drug group and electrical stimulation group (P<0.05). Compared with before treatment, the endometrium MVD values of the IUA patients in electrical stimulation group and combination group after treatment were increased significantly (P<0.05); the endometrium MVD value of the IUA patients in combination group was significantly higher than those in control group, drug group and electrical stimulation group (P<0.05). Compared with before treatment, the expression levels of VEGF mRNA and protein in endometrial tissue of the IUA patients in control group, drug group, electrical stimulation group and combination group after treatment were significantly increased (P<0.05); after treatment, the expression levels of VEGF mRNA and protein of the IUA patients in combination group were significantly higher than those in control group, drug group and electricalal stimulation group (P<0.05). Conclusion: Aspirin combined with electrical stimulation pelvic floor smooth muscle can significantly improve the ET of the patients, and increase the uterine blood circulation after hysterocopic adhesiolysis.

Objective: To analyze the distribution of T lymphocyte subsets in peripheral blood of the systemic lupus erythematosus(SLE) patients, and to clarify its relationships with SLE disease activity and prognosis. Methods: The peripheral blood of 100 patients with primary SLE was collected, the counts of T lymphocyte subsets in the patients with different clinical subtypes of SLE (kidney involvement, skin involvement, joint involvement,hematological system involvement and different disease activity stages) were analyzed, and the relationships between the clinical indexes and the T lymphocyte subsets in the patients with primary SLE at baseline were evaluated.A total of 78 among 100 patients were followed up for 12 months, among them 26 patients had poor prognosis;the counts of T lymphocyte subsets in the patients with SLE were compared between poor prognosis group and clinical remission group. The correlations between the counts of T lymphocyte subsets in the SLE patients and the clinical indexes were analyzed by Spearman correlation test. Results: The counts of CD3+ T lymphocytes, CD4+ T lymphocytes and CD8+ T lymphocytes of the SLE patients in lupus nephritis group were lower than those in non-lupus nephritis group (P<0.05), the counts of CD3+ T lymphocytes and CD4+ T lymphocytes in the SLE patients in joint involvement group were lower than those in non-joint involvement group (P<0.05), the count of CD8+ T lymphocytes of the SLE patients in skin involvement group was lower than that in non-skin involvement group (P<0.05), and the counts of CD3+ T lymphocytes,CD4+ T lymphocytes and CD8+ T lymphocytes of the SLE patients in hematological system involvement group were lower than those in non- hematological system involvement group (P<0.05). The counts of CD3+ T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes of the SLE patients were negatively correlated with the SLE disease activity index(SLEDAI) score (r=-0.391, P=0.001; r=-0.360, P=0.002; r=-0.315, P=0.008); the counts of CD3+ T lymphocytes, CD4+ T lymphocytes,and CD8+ T lymphocytes were positively correlated with the plasma C3 level of the SLE patients (r=0.407, P=0.001; r=0.395, P=0.001; r=0.254, P=0.035); the counts of CD3+ T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes were positively correlated with the lymphocyte (LY) level in peripheral blood of the SLE patients (r=0.717,P=0.000;r=0.617,P=0.000;r=0.599,P=0.000);the counts of CD3+ T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes of the SLE patients in severe activity group were significantly lower than those in mild activity group(P<0.05);the counts of CD3+ T lymphocytes and CD4+ T lymphocytes of the SLE patients in poor prognosis group were lower than those in clinical remission group (P<0.05). Conclusion: The distribution of T lymphocyte subsets of the SLE patients in different subgroups is different, and it is closely associated with the SLE disease activity;the low count of CD4+T lymphocytes is associated with the poor prognosis of SLE patients, which may be a marker of SLE disease activity and prognostic judgment.

Objective: To investigate the feasiblity of 3D printing technology-assisted pre-operative design in guiding the performance of individualized customization elbow joint prosthesis replacement. Methods: A male patient with comminuted fracture of the right distal humerus after operation was selected,the comminuted fracture of the distal humerus was treated with open reduction and internal fixation 10 years ago. Because of internal fixation fracture,the joint appeared stiffness and pain, and the activity was restricted after treatment; the patient was admitted to hospital for elbow joint function recovery and pain relief, and the patient was given the 3D printing technology-assisted individualized customization elbow joint prosthesis replacement. The computer-assisted design technique combined with 3D printing technology was used to simulate the reconstruted anatomly morphology to confirm the osteotomy range;the personalized elbow prosthesis was designed based on the reconstructed anatomy morphology. During the operation, the osteotomy and elbow joint reconstruction were performed according to the design. Results: The operation was successfully completed,and the intraoperative positioning results showed that the elbow joint prosthesis was in good position and achieved the preoperative design effect.The postoperative follow-up result was satisfactory,the activity of elbow joint could satisfy the daily needs of the patient 6 months after operation. Thirty-six months after operation, the HSS score was increased from 15 points before operation to 90 points after operation. Conclusion: For the patients with internal fixation fracture after complicated distal humerus fracture, the computer-aided design technique combined with 3D printing technology can be used to assist the prosthesis design and preoperative design,and the elbow joint anatomy can be reconstructed and the function can be restored by this method.

Objective: To discuss the pathogenesis, clinical manifestation, diagnosis and operation methods of the patients with unilateral inner ear Mondini malformation complicated with cerebrospinal fluid ear leakage, and to improve the understanding of the clinical features. Methods: The clinical materials of one patient diagnosed as unilateral inner ear Mondini malformation complicated with cerebrospinal fluid ear leakage were collected, and the clinical features, imageological performance and diagnosis and treatment methods were analyzed combined with the relevant literatures. Results: Because of left nasal discharge, headache for 20 years, aggravation for 1 month,the patient went to our hospital. The physical examination results showed that the left tympanic membrane was invaginated and intact, and there was hearing loss in the left ear.The endoscopic examination results showed that when the intracranial pressure was increased, the cerebrospinal fluid flowed out from the left eustachian tube orifice. The Mondini malformation and cerebrospinal fluid otorrhea in the left ear were initially considered in combination with the clinical manifestations, and relative examination results of the patient. The left cerebrospinal fluid otorrhea repair was performed, the postoperative hearing was good, and there was no nasal flow, headache and discomfort. The patient was followed up for 1 year and there was no recurrence of meningitis. Conclusion: The patient with recurrently unclear meningitis would be firstly considered as congenital malformation of inner ear. The diagnosis of the disease is mainly based on the examinations of temporal bone CT and MRI; the tymanic exploration and repair surgery might be an effective method,and operation is the main treatment method.

Objective: To analyze the clinical characteristics, diagnostic criteria and treatment methods of the children with degenerative large cell lymphoma (ALCL), and to improve the diagnosis and treatment level of the clinicians. Methods: The clinical data of one child with ALCL were retrospectively analyzed, including clinical symptoms, signs, auxiliary examination, diagnosis basis, differential diagnosis, treatment plan and disease outcome,and the key factors of diagnosis and treatment of the disease were summarized combined with literature review. Results: The child went to hospital because of progressive lymph node enlargement as the first symptom. The lips were red, the epidermis was cleft, the skin was dry, both hands were prominent, and the palm was cleft and the flaky desquamation was visible on the fingertips;multiple swollen lymph nodes were found on the sides of neck, the boundary was unclear, the activity was poor,and tenderness was also found; the tongue nipple presented strawberry tongue. The biopsy pathological results showed ALK-positive ALCL. There were no abnormalities in the bone marrow smear; the abnormal phenotypic T lymphocytes were found in the bone marrow immunotype, peripheral blood immunotype and cerebrospinal fluid immunotype. According to the International Childhood Non-Hodgkin's Lymphoma Staging System (2012),the chlid was clearly diagnosed as ALK-positive ALCL (stage Ⅳ, R3 group).After the induction chemotherapy of APO regimen was given, the condition of the patient was up to partial remission(PR),and the treatment regimen was replaced with B-NHL-BFM-90 regimen; after one cycle of modified AA regimen, the bone marrow and peripheral blood immunotype of the patient turned negative. Conclusion: The clinical manifestations of ALCL patients are different; the lymph node biopsy is the only basis for the diagnosis of ALCL,and hormones could not be blindly used before definitive diagnosis.

Objective: To explore the curative effect of local radiotherapy combined with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in the treatment of the patient with unresectable and low-grade pulmonary mucoepidermal carcinoma (PMEC) with positive EGFR mutation, to analyze the effectiveness and safety of the treatment mode, and to provide the clinical reference for the treatment of this disease. Methods: The clinical data of a patient who diagnosed as PMEC was collected. The relationships between the changes of imaging and clinical symptoms and the prognosis of the patient were analyzed and the relative literatures were reviewed. Results: A 22-year-old woman was admitted to the hospital due to shortness of breath and 2 months of cough.The pathological diagnosis was PMEC,the L858R mutation in exon 21 of EGFR gene was found by gene detection and the tumor could not be resected by surgery.The patient was treated with gefitinib 250 mg·d^-1 orally combined with radiotherapy,and the dose of radiotherapy was 50 Gy totally with 2 Gy per fraction(25 times). After the treatment of local radiotherapy combined with oral administration of gefitinib, the tumor size was shrunk compared with before radiotherapy. The tumor size was shrunk further after the intervention of continuous oral gefitinib;the patient had no adverse reactions except the mild skin rash,and the progression free survival (PFS) was 18 months. Conclusion: For the unresectable and low-grade PMEC patient with positive EGFR mutation, the treatment mode of radiotherapy combined with EGFR-TKIs has good efficacy and the side effects could be accepted.

Objective: To explore the orthodontic results of two-phase treatment of Twin Block appliance and Invisalign appliance in the patient with Angle class Ⅱ divison 1 malocclusion, and to provide the basis for the treatment in clinics. Methods: A patient with mandibular retraction accompanied by crowdedness, who was diagnosed as Angle class Ⅱ divison 1 malocclusion, MaoⅡ²+Ⅳ¹+Ⅰ¹ malocclusion was selected, and the curative effect of this method was discussed combined with the literature review. Results: Twin Block appliance and Invisalign appliance were applied for two-phase treatment, then the lateral cephalometric radiograph, the lengths and widths of the dental arches were measured and the measurement values were compared between before and after treatment;the changes of Ricketts esthetic planes of the patient before and after treatment were analyzed. Compared with before treatment, the angle SNB was increased by 1.7°, the angle ANB was decreased by 0.7°, the angle of L1/MP was increased by 8°, and the angle of U1/L1 was decreased by 6°; the distance of U1-NA was increased, while the distance of L1-NB was decreased;the width of middle maxillary dental arch was increased by 3.8 mm, the width of posterior mandibilular dental arch was increased by 3.4 mm, and the lengths of maxillary and mandibilular dental arches were also increased. The results of Ricketts esthetic plane analysis showed the position of pogonion moved forward after treatment. Conclusion: Twin Block appliance and Invisalign appliance as two-phase treatment can be used for the patients with Angle class Ⅱ divison 1 malocclusion, and it can improve the facial profile of patient;the method can achieve the therapeutic purpose while bringing excellent orthodontic experience to the patients.

Objective: To explore the clinical characteristics,diagnosis and treatment methods of pulomnary sarcoidosis,and to improve the clinicians' understanding of pulmonary sarcoidosis. Methods: The clinical materials,the results of bronchoscope and pathological examinations of one pulomnary sarcoidosis patient were collected,and the relative literatures were reviewed. Results: A patient with cough and progressive dyspnea for 2 months was admitted to the hospital,and the patient had no obvious positive signs. The chest CT images showed diffuse nodules in both lungs, bilateral hilar and mediastinal lymph node enlargement; lung cancer with pulmonary lymphangitic carcinomatosis(PLC),and metastasis of bilateral supraclavicular, bilateral hilar, mediastinal lymph nodes were diagnosed by PET-CT images.The pathological results of fiberoptic bronchoscope after hospitalization showed a chronic granulomatous inflammation in the lung tissue,without obvious necrosis, and tuberculosis could not be entirely excluded.The patients were given glucocorticoids and preventive anti-tuberculosis drugs; 1 month later, the chest CT showed a significant reduction in the bilateral pulmonary nodules; 3 months later, the symptoms of patient disappeared;the chest CT showed the biateral pulmonary nodules basically disappeared,the bilateral hilar and mediastinal lymph nodes were markedly shrunk;the pulmonary sarcoidosis was diagnosed finally. Conclusion: The diagnosis of pulomnary sarcoidosis should be mainly based on the pathological diagnosis combined with clinical manifestation of the patient;sarcoidosis is easily misdiagnosed as tumor or tuberculosis only by the clinical manifestations and the imaging results.

Objective: To analyze the clinical characteristics of one child with acute necrotizing encephalopathy(ANE) with good prognosis, and to improve the clinicians' understandings of the disease. Methods: The clinical materials of a patient with ANE were collected, and the clinical features and diagnosis and treatment experience were retrospectively analyzed combined with the relative literatures. Results: The previously healthy boy,aged 3 years old,was admitted to the hospital due to pneumonia and his condition was improved after anti-infection treatment. On the 4th day, the patient had a high fever again;on the 10th day, the patient suddenly became generalized convulsive seizures, then the patient was in coma. The brain magnetic resonance imaging (MRI) revealed the multiple and symmetric necrosis in the bilateral thalami, brainstem and other areas. The patient was diagnosed as ANE and systemic inflammatory response syndrome (SIRS) and was treated with anti-infection, intravenous high-dose methylprednisolone, intravenous immunoglobulin and symptomatic treatment. After regular outpatient follow-up of 3 months,both Gesell Development and Development of Social Skills assessment of the patient improved to the marginal defect level;the patient was expected to achieve self-care and was still in rehabilitation treatment. Conclusion: The early clinical manifestations of ANE patients are lack of specificity, often accompaning high fever, rapid disease progression and critical condition;the diagnosis of ANE is mainly based on brain MRI findings;early and timely immunomodulatory therapy is effective and can improve the the prognosis of the patients with ANE.

Objective: To constract the mouse models of chronic obstructive pulmonary disease(COPD) induced by intranasal instillation of cigarette smoke extract(CSE) and lipopolysaccharide(LPS),and to explore its feasibility. Methods: Twenty-four C57BL/6J male mice were randomly divided into control group and model group, and there were 12 mice in each group. The COPD mouse model was established by intranasal instillation of CSE and LPS. The number of total cells,neutrophils and macrophages in bronchoalveolar lavage fluid(BALF) of the mice in two groups were determined,the pathological changes of lung tissue of the mice in two groups were observed, and the number of PAS positive cells,alveolar mean lining interval (MLI) and alveolar damage index (DI) (P<0.01)of the mice in two groups were determined.Western blotting method was used to detect the expression levels of E-cadherin, Vimentin, and α-SMA proteins in lung tissue of the mice in two groups. Results: The number of total cells,neutrophilis and macrophages in BALF of the mice in model group at 4 and 6 weeks of experiment was significantly higher than those in control group (P<0.01),and the typical mucus hypersecretion and emphysema in lung tissue of the mice in model group were found; the number of PAS positive cells, MLI(P<0.01),and DI(P<0.01) of the mice in model group at 4 and 6 weeks were higher than those in control group.Compared with control group,the expression level of E-cadherin protein in lung tissue of the mice in model group was decreased(P<0.01), and the expression levels of Vimentin and α-SMA proteins in lung tissue of the mice in model group were increased(P<0.01). Conclusion: The mouse model of COPD is successfully established by intranasal instillation of CSE and LPS in a short period of time.