Objective: To compare the effects of sequential and single-step culture media systems on the development of human early embryo in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles, and to provide a reference for the selection and evaluation of the human embryo culture system of assisted reproductive technology (ART). Methods: A total of 155 patients received IVF/ICSI treatment were selected and the ova from the same patient were divided into two groups. The ova were cultured in Vitrolife sequential media system G-IVF/G1/G2 culture solution(sequential culture group) and Irvine single-step media system CSCC culture solution(single-step culture group)for IVF and embryo culture.The fertilization and early embryo development in the different culture systems in two groups were observed. Results: Compared with single-step culture group, the fertilization rate, 2PN fertilization rate and multi-nuclear fertilization rate of the patients in sequential culture group had no significant differences (P>0.05). In the IVF patients, there were no significant differences in the cleavage rates, quality embryo rates, high quality embryo rates and blastocyst formation rates between sequential culture group and single-step culture group (P>0.05). In the ICSI patients, there were also no significant differences in the cleavage rates, quality embryo rates and blastocyst formation rates between sequential culture group and single-step culture group (P>0.05). However, the high quality embryo rate of the patients in sequential culture group was significantly higher than that in single-step culture group (P=0.015). Both in the IVF and ICSI patients, the percents of densification embryos in single-step culture group were significantly higher than those in sequential culture group (P=0.001). Moreover, the embryos cultured in sequental culture group were smooth and homogeneous, but the embryos cultured in single-step culture group were rough and granular. Conclusion: There are no significant differences in fertilization and early embryo development between the two culture media systems.

Objective: To explore the protective effect of glutamine(GLN) on the hyperoxia-induced lung injuryof the neonatal rats through endoplasmic reticulum stress (ERS) pathway, and to elucidate its mechanisms. Methods: A total of 90 Wistar rats were randomly divided into control group (FiO₂=21%), hyperoxia group(FiO₂>85%), and hyperoxia+GLN group (FiO₂>85%,the concentration of intraperitoneal injection of GLN was 0.75 g·kg^-1·d^-1);there were 30 rats in each group. The body weights and water contents in the lung tissue of the neonatal rats were measured on the 3rd, 7th and 14th days of the experiment. HE staining was used to determine the morphology of lung tissue of the rats. The superoxide dismutase (SOD) activity in lung tissue of the rats was detected by nitro blue tetrazolium chloride(NBT), and the malondialdehyde (MDA) level was determined by thiobarbital acid(TBA). The expression levels of Caspase-12, GADD153, GRP78, Bcl-2,and Bax in lung tissue of the rats were detected by Western blotting method. Results: Compared with control group at the same time, the body weights of the neonatal rats in hyperoxia group on the 3rd,7th and 14th days were significantly decreased(P<0.05), the water contents in lung tissue of the neonatal rats were increased(P<0.05), the SOD activities were significantly decreased(P<0.05), the levels of MDA in the lung tissue of the neonatal rats were increased(P<0.05), the expressions levels of Caspase-12, GADD153, GRP78 and Bax proteins were significantly increased(P<0.05),and the expression levels of Bcl-2 protein and the Bcl-2/Bax ratios were significantly decreased(P<0.05).Compared with hyperoxia group at the same time, the body weights of the neonatal rats in hyperoxia + GLN group on the 3rd,7th and 14th days were significantly increased(P<0.05), the water contents in lung tissue of the neonatal rats were decreased(P<0.05), the SOD activities were significantly increased(P<0.05), the levels of MDA in lung tissue of the neonatal rats were decreased(P<0.05), the expression levels of Caspase-12, GADD153, GRP78 and Bax proteins were significantly decreased(P<0.05), the expression levels of Bcl-2 protein and the Bcl-2/Bax ratios were increased(P<0.05).The pathological sections of lung tissue of the rats in control group showed that lung tissue structure was regular, no alveolar edema was found,the alveolar size and alveolar septum were approximately the same, and no inflammatory cell infiltration was found; the histopathological sections of lung tissue of the rats in hyperoxia group showed swelling of brochial and alveolar epithelial cells, enlargement of alveolar lumen, edema of interstitial cells, inflammatory cell infiltration and fibrous exudation;the degrees of alveolar damage, the inflammatory exudation and the proliferation of fibrons tissue in hyperoxia+GLN group were alleviated which was between hyperoxia group and control group. Conclusion: GLN can alleviate the hyperoxia-induced lung tissue edema and inflammatory response of the neonatal rats, and one of mechanisms is that GLN can down-regulate the expression levels of Caspase-12, GADD153, GRP78 and Bax proteins and up-regulate the expression level of Bcl-2 protein through ERS pathway to protect hypoxic lung injury.

Objective: To detect the levels of angioteinsinⅡ(AngⅡ) and angioteinsin(1-7)[Ang(1-7)] and the expression levels of angiotensin Ⅱ type-1 receptor(AT1R) and Mas receptor (MasR) proteins in kidney tissue of the limb ischemia-reperfusion (LIR) mice pre-treated with the angiotensin coverting enzyme 2(ACE2) activator diminazene(DIZE), and to explore the protective effect of DIZE on the kidney injury of the LIR mice. Methods: Eighteen male ICR mice aged 8 weeks were divided into control group, LIR group and LIR+DIZE group. The mice in model group and LIR+DIZE group were subjected to 2 h of ischemia and 4 h of reperfusion to establish the LIR models. The mice in LIR+DIZE group were pre-treated with 10 mg·kg^-1·d^-1 DIZE for 14 d by subcutaneous injection before LIR. The histological technique was used to observe the morphology of kidney tissue of the mice and the pathological injury was evaluated. Chemical colorimetry was performed to determine the levels of serum urea and serum creatinine(Scr) of the mice. Enzyme linked immunosorbent assay (ELISA)was used to determine the AngⅡand Ang(1-7) levels in kidney tissue of the mice. Western blotting method was used to measure the expression levels of AT1R and MasR proteins in kidney tissue of the mice. Results: Compared with control group,the pathological changes such as inflammatory cell infiltration and epithelial cell degeneration were found in kidney tissue of the mice in LIR group, and the kindey injury score was obviously increased (P<0.05);compared with LIR group, the kidney injury performance in the kidey tissue of the mice in LIR+DIZE group was alleviated and the kidney injury score was decreased significantly(P<0.05).Compared with control group, the levels of serum urea and Scr of the mice in LIR group were significantly increased (P<0.05);compared with LIR group, the levels of serum urea and Scr of the mice in LIR+DIZE group were significantly decreased (P<0.05).Compared with control group, the AngⅡ, Ang(1-7) levels and the ratio of AngⅡ/Ang(1-7) of the mice in LIR group were significantly increased (P<0.05); compared with LIR group, the AngⅡlevel of the mice in LIR+DIZE group was markedly decreased(P<0.05), the Ang(1-7) level was significantly increased(P<0.05)), and the ratio of AngⅡ/Ang(1-7) was decreased(P<0.05).Compared with control group,the expression level of AT1R protein in kidney tissue of the mice in LIR group was significantly decreased(P<0.05), the expression of MasR protein was significantly increased (P<0.05), and the AT1R/MasR ratio was decreased(P<0.05).Compared with LIR group,the AT1R and MasR protein expression levels in kidney tissue of the mice in LIR+DIZE group were significantly increased(P<0.05), and the AT1R/MasR ratio was also increased(P<0.05). Conclusion: The imbalance of AngⅡ/Ang (1-7) and AT1R/Mas expressions in kidney tissue of the mice may be involved in kidney injury after LIR of the mice. ACE2 activitor DIZE may play a protective role in the kidney by improving the imbalance of AngⅡ/Ang(1-7) and AT1R/MasR expressions.

Objective: To detect the expressions of lncRNA H19(H19) and IL-6/STAT3 pathway in the ulcerative colitis-associated colorectal cancer(CAC) tissue of the mice, and to explore its possible mechanism. Methods: A total of 22 C57BL/6 mice were randomly divided into control group(n=10) and model group(n=12). The CAC models were induced by azomethane(AOM) combined with dextran sodium sulfate(DSS) in the mice in model group. The mice were sacrificed on the 120th day, the disease activity index(DAI) of the mice was evaluated, the tumor formation rate was evaluated,the colon length was measured, and the pathomorphology of colon tissue of the mice was observed by HE staining. The serum IL-6 level of the mice was detected by ELISA.The expression levels of H19, let-7a, IL-6, STAT3 and c-Myc mRNA in colon tissue of the mice were detected by qPCR method.The expression levels of p-STAT3 and c-Myc proteins in colon tissue of the mice were detected by Western blotting method. Results: Compared with control group, the tumor formation rate of the mice in model group was 100%, the colon length was significantly shortened (P<0.01), the DAI score was increased (P<0.01), the colon tissue showed the intraepithelial neoplasia by HE staining,the expression levels of H19, IL-6, STAT3 and c-myc mRNA in colon tissue were significantly increased (P<0.01), the expression level of let-7a mRNA in colon tissue was significantly decreased (P<0.01), the serum IL-6 level was increased (P<0.01), and the expression levels of p-STAT3 and c-Myc proteins in colon tissue were increased (P<0.01). Conclusion: The pathogenesis of CAC in the mice may be related to the up-regulation of c-Myc and H19 and down-regulation of let-7a, which are mediated by IL-6/STAT3 pathway.

Objective: To observe the effect of neuroopilin-1(NRP1) gene on the process of radiation-induced pulmonary fibrosis(RIPF), and to explore its roles in the occurrence and development of epithelial-mesenchymal transition(EMT) mediated by Wnt/β-catenin pathway tail identification was performed in and TGF-β1/Smads pathway, and extracellular matrix (ECM) deposition. Methods: The Cre-LoxP recombinase system was used to construct the transgenic C57BL/6J mice with NRP1 gene specific knockout in alveolar type Ⅱ epithelial cells(AT-Ⅱ) and the mice. A total of 160 mice were randomly divided into 4-week group, 8-week group, 16-week group and 24-week group. In each group, the mice were randomly divided into wild type (Con) group, wild type+irradiation (IR) group, NRP1 gene-specific knockout (KO-Con) group, NRP1 gene-specific knockout+irradiation (KO+IR) group according to the method of random number table;there were 10 mice per group. In KO-Con and KO+IR groups, the NRP1 gene was specifically knocked out in the AT-Ⅱ cells by intraperitoneal injection of tamoxifen, and the mouse models of RTPF were established by 20 Gy total thoracic irradiation in IR group and KO+IR group. After the models were constructed, HE staining and Masson staining were used to verify whether the models were successfully constructed. Immunohistochemistry (IHC) method was used to detect the type Ⅰ collagen (Col Ⅰ) and α-smooth muscle actin (α-SMA) protein expression levels; Western blotting method was performed to detect the NRP1, β-catenin, TGF-β1,and Smad2 protein expression levels in the lung tissue of the mice; Quantitative fluorensence real-time polymerase chain reaction (qRT-PCR) method was used to detect the expression levels of NRP1, Col Ⅰ, α-SMA, β-catenin, TGF-β1, Smad2, E-cadherin, N-cadherin, and Vimentin mRNA in the lung tissue of the mice. Results: The results of HE and Masson staining showed the RTPF models were suceessfully established,and the lung tissue of the mice in IR group mainly showed the pathomorphology of radiation pneumonitis. Compared with Con group, the protein and mRNA expression levels of NRP1 in the lung tissue of the mice in IR group were gradually increased with the prolongation of time(P<0.05), and reached the highest at 24 weeks (P<0.01).Compared with Con group, the expression levels of Col Ⅰ,α-SMA, β-catenin, TGF-β1, and Smad2 proteins and mRNA in the lung tissue of the mice in IR group and KO+IR group were increased gradually with the prolongation of time (P<0.05 or P<0.01).Compared with IR group, the expression levels of Col Ⅰ,α-SMA, β-catenin, TGF-β1, and Smad2 protein and mRNA in the lung tissue of the mice in KO+IR group were significantly decreased(P<0.05 or P<0.01), but they were higher than those in Con group(P<0.05 or P<0.01).Compared with Con group, the expression levels of the epithelial cell marker E-Cadherin mRNA in the lung tissue of the mice in IR group and KO+IR group were gradually decreased with the prolongation of time(P<0.01), and the expression levels of the interstitial cell markers N-Cadherin and Vimentin were increased (P<0.05 or P<0.01), but the expression levels of E-cadhern mRNA in the lung tissue of the mice in KO-IR group were significantly higher than those in IR group(P<0.05 or P<0.01),and the expression levels of N-Cadherin and Vimentin mRNA in the lung tissue of the mice in KO+IR group at each time point were lower than those in IR group (P<0.05or P<0.01). Conclusion: Knockout of NRP1 gene can inhibit the occurrence and development of RTPF, and its mechanism may be involved in regulating the expressions of Wnt/β-catenin and TGF-β1/Smads signaling pathways in the lung tissue and inhibiting the EMT process in the mice.

Objective: To investigate the repair effect of cannabinoid (WIN55212-2) on the myelin sheath of the demyelination model mice induced by cuprizone, and to elucidate its possible mechanism. Methods: A total of 95 C57BL/6 mice were randomly divided into blank control group, model group (0.2% cuprizone for 6 weeks),DMSO group (intraperitoneally injected with the same amount of DMSO mixture from the 3rd week of feeding 0.2% cuprizone) and treatment group (intraperitoneally injected with 1 mg·kg^-1 WIN55212-2 from the 3rd week of feeding 0.2% cuprizone). The myelin sheath tissue of the mice was stained by Black-gold Ⅱ staining technique, and its histomorphology was observed. The expression levels of juxtanodin(JN),myelin basic protein(MBP) and Nkx2.2 proteins in mouse brain tissue were detected by Western blotting method. Results: The results of Black-goldⅡstaining showed that the corpus callosum of the brain of the mice in blank control group had significant coloration, while the corpus callosum of the mice in model group showed less coloration.Compared with blank control group, the expression levels of JN and MBP proteins in brain tissue of the mice in model group were significantly decreased(P<0.05).Compared with model group and DMSO group, the expression level of JN and Nkx2.2 proteins in brain tissue of the mice in treatment group were significantly increased(P<0.05 or P<0.01). Conclusion: Cannabinoid may promote the JN expression in brain tissue of the cuprizone-induced demyelinating model mice through the transcription factor Nkx2.2, and further promote the remyelination and repair.

Objective: To establish the experimental peri-implantitis mouse models,to investigate the expression of tumor necrosis factor-α (TNF-α) in the gingival tissue of the experimental peri-implantitis model mice, and to elucidate its pathogenic effect on peri-implantitis. Methods: Eighteen 4-week-old C57BL/6J male mice were divided into blank group,control group and model group(n=6); the right and upper first molars were removed, and the custom implants were immediately implanted. After 4 weeks of healing, the mice in model group were used to establish the peri-implantitis models with ligation method; the mice in blank group and control group didn't receive any treatment. The mice in blank group were killed on the 0th day of ligation (before the ligation treatment); the mice in control group were killed on the second week without ligation;the mice in model group were killed on the second week of ligation of the neck of the implant with 5-0 silk thread.The upper jaw bone and gingvival tissue samples of the mice in various groups were obtained,and the morphology of peri-implant gingival tissue were observed with surgical microscope. Micro-CT scanning was used to measure the bone height and bone density around the implants and RT-PCR method was performed to detect the expession level of TNF-α mRNA in the gingival tissue. Results: The morphological observation showed that the gingival tissue around the implants in model group appeared obvious redness, edema, softening and exudation, while there were no visible signs of inflammation in blank group and control group. Compared with control group, the proximal, distal, buccal and zygomatic bone heights and the alveolar bone dellsity around the implants of the mice in model group were significantly reduced (P<0.05), and the expression level of TNF-α mRNA in the gingival tissue of the mice in model group were significantly increased (P<0.05). Compared with blank group, the proximal, distal, buccal and zygomatic bone heights and the alveolar bone density around the implants of the mice in control group had no statistical differences (P> 0.05), and the expression level of TNF-α mRNA in the gingival tissue of the mice in control group had no statistical difference (P> 0.05). Conclusion: An experimental mouse model of peri-implantitis is successfully established with ligation-inducing method. TNF-α is highly expressed in the gingival tissue of the model mice, which may have a role in promoting the pathogenesis of peri-implantitis.

Objective: To investigate the effect of silencing sirtuin 3 (Sirt3) on the apoptosis of human ovarian cancer SKOV3 cells induced by resveratrol (Res),and to explore its mechanism of promoting apoptosis. Methods: The human ovarian cancer SKOC3 cells were cultured with different concentrations (0, 2.5, 5.0, 10.0, 20.0, 40.0 and 80.0 mg·L^-1) of Res for 24 h. The survival rate of cells was measured by MTT assay. The SKOV3 cells were randomly divided into control group,Sirt3 inhibitory 3-(1H-1,2,3-triazol-4-yl)pyridine(3-TYP group, Res group and 3-TYP+Res group. After 24 h of culture, the inhibitory rates of proliferation of the cells in various groups were detected by MTT assay; the nuclei were stained with Hoechst 33342, and the morphorgy nucleus was observed by laser confocal microscope; reactive oxygen species(ROS)probe was used to detect the intracellular ROS levels; Western blotting method was used to detect the expression levels of Sirt3, Bax, Bcl-2 and cleaved caspase-3 proteins in the cells in various groups. Results: The results of MTT assay showed that the survival rates of SKOV3 cells were significantly decreased with the increase of concentration of Res, and the median inhibitory concentration(IC₅₀)was 42.73 mg·L^-1. Compared with control group, the inhibitory rates of proliferation of cells in Res group and 3-TYP+Res group were significantly decreased (P<0.05);compared with Res group,the inhibitory rate of proliferation of the SKOV3 cells in 3-TYP +Res group was significantly inereased(P<0.01). Compared with control group, the nucleus of SKOV3 cells in 3-TYP+Res group showed pyknosis, enhanced staining and more nuclear fragmentation. Compared with control group, the red fluorescence of the SKOV3 cells in 3-TYP group had no significant change, and the red fluorescence of the SKOV3 cells in Res group and 3-TYP+Res group was significantly decreased. The results of Western blotting method showed that compared with control group,the expression level of Sirt3 protein in the cells in 3-TYP group was significantly decreased (P<0.05),and the expression levels of Bcl-2, Bax and cleaved caspase-3 in the cells did not change significantly(P>0.05);the protein expression levels of Sirt3 and Bcl-2 proteins in Res group were significantly decreased (P<0.05), and the expression levels of Bax and cleaved caspase-3 proteins were significantly increased (P<0.05).Compared with Res group, the expression levels of Bax and cleaved caspase-3 proteins in 3-TYP+Res group were significantly increased (P<0.05), and the expression levels of Bcl-2 and Sirt3 proteins in 3-TYP+Res group were significantly decreased (P<0.05). Conclusion: Res can induce the apoptosis of SKOV3 cells, and the inhibition of Sirt3 expression by 3-TYP can enhance the effect of Res.

Objective: To observe the effect of Schisandra Chinensis polysaccharide (SCP) on the serum inflammatory factors in the rats with type 2 diabetes mellitus (T2DM) induced by high-fat diet and lowdose of streptozotocin (STZ), and to explore its underlying mechanism in the treatment of T2DM. Methods: The male Wistar rats were given high-fat diet and introperieoneally injected with low dose of STZ (30 mg·kg^-1) in one time to establish the rat T2DM models. The successful model rats were randomly divided into model group, low dose (25 mg·kg^-1) of SCP group, middle dose (50 mg·kg^-1) of SCP group and high dose (100 mg·kg^-1) of SCP group; there were 10 rats in each group. Another 10 healthy rats were used as normal control group. Eight weeks after the intragastric administration of SCP, oral glucose tolerance test (OGTT) was performed in the rats of various groups. The fasting blood glucose (FBG) and insulin (INS) levels were detected by glucose oxidase method and radioimmunoassay method, respectively, and the insulin sensitivity index (ISI) was calculated. The levels of interleukin-6 (IL-6), C-reactive protein (CRP), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) in serum of the rats were measured by enzyme linked immunosorbent assay (ELISA) method. HE staining was used to observe the pathomorphology of pancreas tissue of the rats. Results: Compared with normal control group, the serum FBG level of the rats in model group was significantly increased (P<0.01), and the area under the curve(AUC) of blood glucose of the rats was significantly increased, the serum INS level and the ISI were significantly decreased (P<0.05 or P<0.01); the levels of IL-6, CRP, IL-1β, TNF-α, and NF-κB in serum were all significantly increased (P<0.05 or P<0.01). Compared with model group, the serum FBG levels of the rats in different doses of SCP groups were markedly decreased (P<0.05), the AUC of blood glucose of the rats were significantly decreased, the INS and the ISI levels were significantly elevated (P<0.05); the levels of IL-6, CRP, IL-1β, TNF-α and NF-κB in serum were significantly decreased (P<0.05 or P<0.01). Compared with low dose of SCP group, the FBG levels of the rats in middle and high doses of SCP groups were significantly decreased(P<0.05),the serum INS level of the rats in high dose of SCP group was significantly increased(P<0.05). The HE staining results showed that compared with normal control group, the islets were atrophied, the number of cells in islets was decreased and the boundary was irregular in model group; compared with model group,the islet boundary in different doses of SCP groups became clear, the areas were increased, and the number cells was increased significantly. Conclusion: SCP can decrease the FBG level, increase the INS level and improve insulin resistance (IR) in the T2DM rats induced by high-fat diet combined with low dose of STZ, and its mechanism might be related to its inhibiting inflammation response.

Objective: To investigate the expression of vascular endothelial cell growth factor (VEGF) in the immediate replantation of pulp healing in the rats, and to clarify the effect and its mechanism of erythropoietin (EPO) on immediate pulp reconstruction in the rats. Methods: Eighty 4-week-old healthy male Wistar rats were randomly divided into non-tooth extraction group, negative control (normal saline) group, positive control (gentamicin) group and EPO group;there were twenty rats in each group.The teeth in each group were immersed in its corresponding solution for 4 min before replantation. Four rats were killed on the days 3, 7, 14, 21 and 28, respectively, and the specimens were made in the operation area. HE staining was used to observe the pulp revascularization in different time periods. Immunohistochemical staining was used to observe the protein expression levels of VEGF in odontal tissue of the rats in each group in different time periods. Results: The HE staining results showed that compared with non-tooth extraction group, the pulp tissue of replanted teeth of the rats in normal saline group had more inflammation, less root development, less restorative dentin and cementum deposition, and wider apical pores; the inflammation of pulp tissue of replanted teeth of the rats in gentamicin group and EPO group was mild, and the root development was relatively good; there were more deposits of restorative dentin and cementum, and the apical pores were narrowed. The immunohistochemical results showed that compared with non-tooth extraction group, the positive expressions of VEGF in odontal tissue of the rats at the days 3, 7 and 14 in the other groups were strong. Afterwards, the positive expression levels of VEGF were decreased gradually with the prolongation of time. The average optical density (AOD) of VEGF positive area indicated that EPO group > gentamycin group > normal saline group > non-tooth extraction group. Compared with non-tooth extraction group, the protein expression levels of VEGF in odontal tissue of the rats in normal saline group, gentamicin group and EPO group at 3, 7, 14 and 21 d after operation were significanty increased (P<0.05), but the protein expression levels of VEGF in odontal tissue of the rats at 28 d had no significant differences (P>0.05). Compared with normal saline group, the protein expression levels of VEGF in odontal tissue of the rats in gentamicin group and EPO group at 3, 7, 14 and 21 d after operation were significantly increased (P<0.05), but the protein expression levels of VEGF in odontal tissue of the rats at 28 d had no significant differences (P>0.05).Compared with gentamicin group,the protein expression levels of VEGF in odontal tissue of the rats in EPO group at every time points had no significant differences(P>0.05). Conclusion: EPO can increase the expression of VEGF, induce angiogenesis in pulp tissue, and provide the rich vascular bed for replantated teeth, so as to exert the potential of dental pulp defense and repair, and promote the healing of replanted teeth.

Objective: To investigate the inhibitory effect of foodborne procyanidins on the growth of human neuroblastoma SH-SY5Y cells, and to elucidate its mechanism. Methods: The SH-SY5Y cells were cultured and divided into control group, 10 mg·L^-1 foodborne procyanidins group, 20 mg·L^-1 foodborne procyanidins group and 40 mg·L^-1 foodborne procyanidins group;the medium containing different concentrations (0, 10, 20 and 40 mg·L^-1) of foodborne procyanidins was added into each group. The proliferation rates of SH-SY5Y cells were measured by MTT method at 24, 48 and 72 h after the drug treatment. Flow cytometry was used to detect the cell cycle of SH-SY5Y cells at 72 h after the drug treatment, and the apoptotic rate of SH-SY5Y cells was detected by Annexin Ⅴ apoptotic assay kit at 72 h after the drug treatment. Results: Compared with control group, the proliferation rates of SH-SY5Y cells at 24, 48 and 72 h in 10, 20 and 40 mg·L^-1 foodborne procyanidins groups were decreased; there were significant differences at 48 and 72 hours in 20 mg·L^-1 foodborne procyanidins group (P<0.05 or P<0.01);there were also significant differences at 24, 48 and 72 h in 40 mg·L^-1 foodborne procyanidins group (P<0.01). Compared with control group, the percentage of cells in G₀/G₁ phase in 40 mg·L^-1 foodborne procyanidins group were increased(P<0.01) and the percentage of cells in G₂/M phase were decreased (P<0.01). Compared with control group, the apoptotic rates of cells in 10, 20 and 40 mg·L^-1 foodborne procyanidins groups were increased significantly (P<0.01). Conclusion: Foodborne procyanidins can inhibit the growth of human neuroblastoma SH-SY5Y cells, and its mechanism is mainly to block the cell cycle and induce the apoptosis.

Objective: To induce the differentiation of bone marrow mesenchymal stem cells(BMSCs) by three culture methods in vitro, and to explore the best method of inducing the BMSCs to differentiate into the cardiomyocytes in vitro. Methods: The rat BMSCs were separated and cultured with density gradient centrifugation combined with adherence culture method.The rat BMSCs were cultured with 5-azacytidine(5-aza), the myocardial cell cleavage of the dilated cardiomyopathy(DCM) model rats and the serum of DCM model rats+5-aza in vitro,respectively; control group was set up at the same time(The BMSCs were cultured in the L-DMEM medium containing 10% FBS under the same condition).The morphology of BMSCs were observed and the expression of troponin T(cTnT) was detected by RT-PCR, Western blotting method and immunohistochemical staining. Results: The rat BMSCs were obtained by density gradient centrifugation combined with adherence culture method. The BMSCs highly expressed the characteristic surface marker CD44, CD29 and CD105 of mesenchymal stem cells (MSCs), and did not express the characteristic surface marker of hematopoietic stem cells CD34. Under the specific induction conditions, the BMSCs were differentiated into the osteoblasts and the aliphatic cells. In vitro, three culture methods could induce the BMSCs to express cTnT and differentiate into the cardiomyoid cells. In 5-aza group, the cell growth status was poor, and the cells in the other two groups had better growth status. The positive expression rate of cTnT was the highest in DCM model rat serum +5-aza group. Conclusion: The effect of DCM model rat serum combined with 5-aza to induce the BMSCs to differentiation into the myocardial cells is the best.

Objective: To study the effect of Matrilin-4 on the reparative dentin formation in the rats after dental pulp injury, and to explore its possibility to be used as a new capping agent. Methods: A total of 28 male Wistar rats were selected. A cavity on the occlusal surface of maxillary first molars on both sides was prepared in each rat. Matrilin-4 was applied on the left exposed pulp(Matrilin-4 group), and PBS was applied on the right exposed pulp(PBS group);and the cavities in both sides were covered with glass ions. On days 3, 7, 14 and 28 after operation, the rats were sacrificed,and the maxillary first molars on both sides of the rats were selected. HE staining and immunohistochemistry staining were used to observe and analyze the reparative dentin formation and the expression of dentin sialoprotein(DSP) in the odontoblasts. Results: The HE staining results showed that there were less inflammatory cells under the exposed pulp in that Matrilin-4 group at 3 d after operation compared with PBS group, and obvious vasodilation was found. At 7 d after operation, the inflammatory reaction under the exposed pulps in two groups was aggravated, but the inflammatory reaction in Matrilin-4 group was significantly lighter than that in PBS group, and the prophase dentin formation was found. After injury of 14 d,the pulp-exposed areas were covered by more complete dentin bridge in Matrilin-4 group; at 28 d after operation, compared with PBS group, there was a thick, complete and reparative dentin bridge under the exposed pulps, composed of tubular dentin in Matrilin-4 group, and there were reorganized odontoblastic layers beneath the dentin bridge. The results of immunohistochemistry showed that the positive expression strengths of DSP in the odontoblasts of the rats in two groups were gradually increased at 3, 7 and 14 d after operation, and were decreased at 28 d. The positive expression strengths of DSP in the odontoblast of the rats in Matrilin-4 group were stronger than those in PBS group at each time point after operation, and the positive expression strength reached the peak at 14 d. Conclusion: Compared with PBS group, the positive expression strength of DSP in the odontoblasts of the rats in Matrilin-4 group had significant differences (P<0.01) at 3, 7 and 14 d.

Objective: To expore the effects of cucurbitacin B(CUB) combined with oxaliplatin(OXA)on the proliferation and apoptosis of human colon cancer SW480 cells, and to clarify their mechanisms. Methods: The SW480 cells were divided into control group, 10,20, and 40 μmol·L^-1 CUB groups, OXA group(100 μmol·L^-1) and combination group (40 μmol·L^-1 CUB +100 μmol·L^-1 OXA). The proliferation rate of the SW480 was determined by MTT assay. Hoechst33258 staining was used to observe the morphology of SW480 cells. The cell cycle and apoptotic rates of SW480 cells were detected by flow cytometry. The expressions levels of caspase-3,cleaved caspase-3,Bax and Bcl-2 proteins were measured by Western blotting method. Results: The MTT results showed that compared with control group, the proliferation rates of the SW480 cells in different doses of CUB groups and OXA group were significantly decreased(P<0.05); compared with different doses of CUB groups and OXA group, the proliferation rate of the SW480 cells in combination group was significantly decreased(P<0.05). The results of Hoechst 33258 staining showed that the blue fluorescence in the SW480 cells in different doses of CUB groups and OXA group were brighter than that in control group,which showed granular blue fluorescence in the cell nucles. Compared with different doses of CUB groups and OXA group, the blue fluorescence in the SW480 cells in combination group was more significantly bright, and granular blue fluorescence was significantly increased. The cell cycle detection results of flow cytometry showed that compared with control group, the percentages of the SW480 cells in G₂/M phase in different doses of CUB groups and combination group were incresaed (P<0.05); the percentages of SW480 cells in S phase in 40 μmol·L^-1 CUB group, OXA group and combination group were incresaed (P<0.05). The apoptosis detection results of flow cytometry showed that compared with control group, the apoptotic rates of the SW480 cells in different doses of CUB groups and OXA group were increased (P<0.05); compared with different doses of CUB groups and OXA group, the apoptotic rate of the SW480 cells in combination group was significantly increased(P<0.05).The Western blotting results showed that compared with control group, the expressions levels of caspase-3 and Bcl-2 in the SW480 cells in different doses of CUB groups and OXA group were decreased (P<0.05), the expression levels of cleaved caspase-3 and Bax were increased (P<0.05),and the Bcl-2/Bax ratios were decreased(P<0.05); compared with different doses of CUB groups and OXA group, the changes of the above protein expression levels,the cleaved caspase-3/caspase-3 ratio and the Bcl-2/Bax ratio in combination group were more obvious (P<0.05). Conclusion: CUB combined with OXA can effectively inhinbit the proliferation of colon cancer SW480 cells, and its mechanism may be related to the S and G₂/M phase arrest,up regulation of the ratio of cleaved-caspase-3/caspase-3 and down-regulation of the ratio of Bcl-2/Bax.

Objective: To investigate the effect of miR-125b on the ventricular remodeling after myocardial infarction in the rats via phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway,and to elucidate itsmechanism. Methods: A total of 75 rats were divided into sham operation group, myocardial infarction group and miR-125b inhibitor group, with 25 rats in each group.The acute myocardial infarction models were established in the rats in myocardial infarction group and miR-125b inhibitor group.The rats in miR-125b inhibitor group was injected with miR-125b inhibitor via the tail vein.The rats were sacrificed 4 weeks later,the body weights were recorded;the hearts were obtained,and the big vessel and left and right auricular appendix were cut out;the heart weights were weighed.The heart weight/body weight (HW/BW), (left ventricle + ventricular septum) weight/body weight (LV+S)/BW, (left ventricular+ventricular septum) weight/heart weight (LV+S)/HW were calculated. HE staining was used to observe the morphological changes of myocardium tissue. Masson staining was used to observe the myocardial fibrosis,and the collagen volume integral of myocardium was calculated.Immunofluorescence staining was used to observe the expression levels of type Ⅰ collagen and type Ⅲ collagen proteins in the myocardium tissue of the rats. Western blotting method was used to detect the expression levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), PI3K, Akt and phosphorylated Akt (p-Akt) in the peripheral region of myocardial infarction of the rats. Results: The HE staining results showed that the myocarclial cells in sham operation group were arranged neatly;the myocardial cells in myocardial infaction group had hypertrophy and disordered arrangement; the myocardial cells in miR-125b inhibitor group were arranged neatly.Compared with sham operation group,the HW/BW, (LV+S)/BW and (LV+S)/HW of the rats in myocardial infarction group were elevated (P<0.05),the collagen volume integral was increased (P<0.05),the protein expression levels of type Ⅰ collagen and type Ⅲ collagen were increased (P<0.05),the expression levels ANP and BNP proteins were increased (P<0.05), and the expression levels of PI3K and p-Akt proteins were decreased (P<0.05).Compared with myocardial infarction group,the HW/BW, (LV+S)/BW and (LV+S)/HW of the rats in miR-125 b inhibitor group were decreased (P<0.05), the collagen volume integral was decreased (P<0.05),the expression levels of type Ⅰ collagen and type Ⅲ collagen proteins were decreased (P<0.05),the expression levels of ANP and BNP proteins were decreased (P<0.05), and the expression levels of PI3K and p-Akt proteins were increased(P<0.05). Conclusion: Inhibition of miR-125b can inhibit ventricular remodeling after myocardial infarction in the rats by inhibiting the PI3K/Akt signaling pathway,and plays an important role in reversing ventricular remodeling after myocardial infarction

Objective: To investigate the effects of anemoside B4 on the blood oxygen stress,inflanmation state and the expressions of apoptosis-related proteins in kidney tissue of the rats with chronic renal failure(CRF), and to illuminate the mechanism of anemoside B4 in protecting the kidney tissue of the CRF rats. Methods: A total of 75 SD male rats were divided into control group, model group, positive control group, low dose of anemoside B4 group and high dose of anemoside B4 group.The CRF rat models were established by continuous administration of adenine for 21 d. After successful modeling, the rats in low and high doses of anemoside B4 groups were given 1 and 2 mg·kg^-1 anemoside B4 by gavage and the rats in positive control group were given 0.5 mg·kg^-1 dexamethasone,lasted for 28 d; the rats in control group and model group were given the same amount of normal saline. Biochemical analyzer was used to detect serum urine creatinine (Scr) and urea nitrogen (BUN) levels. The kit method was used to detect the activity of superoxide dismutase (SOD) and the levels of malondialdehyde (MDA) in kidney tissue of the rats. ELISA was used to detect the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factro-α (TNF-α) in kidney tissue of the rats. Western blotting method was used to detect the activation of PI3K/Akt pathway and the expression levels of apopotosis-related proteins Bcl-2, Bax and Caspase-3 in kindey tissue. Results: Compared with control group,the serum Scr and BUN levels of the rats in model group were increased(P<0.01),the number of normal glomerulus and renal corpuscles were decreased,the activity of SOD in kidney tissue was decreased(P<0.01),the levels of MDA,IL-1β,IL-6 and TNF-α were increased(P<0.01),the expression levels of Bax and Caspase-3 proteins were increased(P<0.01),and the expression levels of p-Akt and Bcl-2 proteins were decreased(P<0.01).Compared with model group, the serum Scr and BUN levels of the rats in positive control group, low and high doses of anemoside B4 groups were significantly decreased (P<0.01), the number of normal glomerulus and renal corpuscles were increased significantly, the activities of SOD in kidney tissue were increased significantly (P<0.01),the levels of MDA, IL-1β, IL-6, and TNF-α were significantly decreased (P<0.01), the protein expression levels of Bax and Caspase-3 were decreased significantly (P<0.01), and the expression levels of p-Akt and Bcl-2 proteins were increased significantly (P<0.01). Conclusion: Anemoside B4 can protect the renal tissue injury by reducing the levels of Scr and BUN in serum, reducing oxidative stress response, reducing the release of inflammatory factors, and activating the PI3K/Akt pathway of renal tissue.

Objective: To discuss the proliferation inhibition and apoptosis induction of shikonin on the FMS-like tyrosine kinase-3 receptor internal tandem duplication (FLT3-ITD) mutated acute myeloid leukemia (AML) MV4-11 cells, and to preliminarily clarify the molecular mechanisms. Methods: The MV4-11 cells were divided into DMSO group and different concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 μmol·L^-1) of shikonin groups, and treated for 24 and 48 h. The inhibitory rate of proliferation was analyzed by CCK-8 assay, and half inhibitory concentration (IC₅₀) was calculated. The MV4-11 cells were divided into blank control group, DMSO group, and different concentrations (0.25, 0.50, and 1.00 μmol·L^-1) of shikonin groups, and treated for 48 and 72 h; the proliferation rate of cells was analyzed by carbox fluorescenceindiacetate succinimidyl este (CFSE). The MV4-11 cells were divided into DMSO group and different concentrations (0.702, 1.404, and 2.808 μmol·L^-1) of shikonin groups, and treated for 48 h;the apoptotic rate was determined by flow cytometry. The MV4-11 cells were divided into DMSO group and different concentrations (0.351, 0.702, and 1.404 μmol·L^-1) of shikonin groups, and treated for 48 h; the microRNA-155 (miR-155) expression level was detected by Real-time PCR. Results: The results of CCK-8 and CFSE methods indicated that the inhibitory rates of proliferation of MV4-11 cells in different concentrations of shikonin groups were increased compared with DMSO grpup(P<0.05 or P<0.01), and the proliferation rates were decreased (P<0.05 or P<0.01) in a concentration-dependent manner; the IC₅₀ of 24 and 48 h were 1.743 and 1.404 μmol·L^-1, respectively. The flow cytometry results showed that the apoptotic rates of the cells in different concentrations of shikonin groups were increased compared with DMSO group (P<0.01) in a concentration-dependent manner. The Real-time PCR results showed that the expression levels of miR-155 in the cells in different concentrations of shikonin groups were decreased significantly(P<0.01), and the expression level in 1.404 μmol·L^-1 shikonin group was decreased by more than 75%. Conclusion: Shikonin could inhibit the proliferation and promote the apoptosis of FLT3-ITD mutated AML MV4-11 cells, and down-regulate the expression of miR-155, suggesting that shikonin may be one of the potential therapeutic drugs for FLT3-ITD mututed AML.

Objective: To measure and anlyze the morphological indexes of talus of the Chinese population with three-dimensional measurement method and to provide the accurate data of morphological parameters of talus, and to pvovide basis for designing the talar prostheses. Methods: Forty male and 40 female volunteers applied for this study and 4 of them were excluded. At last, 76 volunteers of 39 males and 37 females were included. The ankle joints of 76 subjects were scanned by CT and the CT images were reconstructed to three-dimensional model with Mimics software.A total of 13 indexes were measured including talar length, breadth, height, and volume, length, breadth, and radian of medial and lateral malleolus articular surfaces, anterior, middle, and posterior breadth of trochlea; among 13 indexes, 9 indexes were measured through Mimics software,and the other 4 indexes were measured through Magics software. Results: All the indexes of the subjects were normally distributed. There were no significant differences in the morphological indexes between left and right talus in either males or females (P>0.05). The talar length, breadth, and height in the males were (60.85±2.82), (42.96±2.59), and (33.76±1.73) mm, respectively, and in the females they were (54.41±2.49), (39.76±1.78), and (29.72±1.20) mm, respectively. Most of the indexes were larger in the males than the females (P<0.05), whereas radian of medial malleolus articular surface was larger in the females than the males (P<0.05).The radian of lateral malleolus articular surface and the posterior breadth of trochlea of the males and the females had no significant differences (P>0.05). Conclusion: CT measurement method to measure the morphological indexes of talus is more convenient and fast,its measurement error is small,and its erpeatability is high.Left and right sides of talus show strong degree of symmetry, thus the morphological indexes of the contralateral talus can be adopted as a reference for designing talus prosthesis.

Objective: To observe the expression changes of insulin-like growth factor 1(IGF-1)and mammalian target of rapamycin(mTOR)in the peripheral blood of patients with advanced non-small cell lung cancer (NSCLC) treated with chemotherapy combined with metformin, and to elucidate its mechanism. Methods: Sixty patients with NSCLC were divided into chemotherapy group (15 cases of adenocarcinoma and squamous cell carcinoma, treated with chemotherapy alone) and combination group (15 cases of adenocarcinoma and squamous cell carcinoma, treated with chemotherapy combined with metformin). The expression levels of IGF-1 and mTOR protein and mRNA in peripheral blood of the patients in two groups were detected by ELISA and quantitative real-time PCR(QT-PCR) method. Pearson univariate analysis and multivariate logistic regression analysis were used to analyze the influencing factors of the treatment of patients with advanced NSCLC;the curative effect was comprehensively evaluated. Results: Compared with chemotherapy group, the differences of the levels of IGF-1 and mTOR and the mRNA expression levels of IGF-1 and mTOR of the patients in combination group before and after treatment were decreased (t=-3.207, P=0.003; t=2.414, P=0.019; t=-3.635, P=0.001; t=-3.737, P=0.001). In adenocarcinoma, compared with chemotherapy group, the differences the levels of IGF-1 and mTOR and the mRNA expression levels of IGF-1 and mTOR of the patients in combination group before and after treatment were decreased (t=5.270, P<0.01; t=2.816, P=0.009; t=-2.621, P=0.019; t=4.039, P<0.01); in squamous cell carcinoma, compared with chemotherapy group, the differences of the levels of IGF-1 and mTOR and the mRNA expression levels of IGF-1 and mTOR in peripheral blood of the patients in combination group before and after treatment were reduced (t=4.164, P<0.01; t=2.670, P=0.012; t=3.072, P=0.008; t=3.502, P=0.002). From the level of disease control rate, the total effective rate of the patients in combination group (80.0%) was significantly higher than that in chemotherapy group (53.3%) (P<0.05).The univariate analysis results showed that gender, smoking, tumor stage, and lymph node metastasis were related to the therapeutic effects of the patients in two groups (P<0.05);the ECOG score and tumor differentiation degree were related to the therapeutic effect of the patients in chemotherapy group (P<0.05),and pleural effusion was related with the therapeutic effect of the patients in combination group (P<0.05). The results of multivariate analysis showed that smoking and tumor stage were the independent risk factors of the patients in two groups (P<0.05),and smoking and lymph node metastasis were the independent risk factors of the patiens in chemotherapy group(P<0.05); smoking, differentiation, and pleural effusion were the independent risk factors of the patients in combination group (P<0.05).Compared with chemotherapy group, the incidences of bone marrow suppression, gastrointestinal reactions, nephrotoxicity, and liver damage of the patients in combination group had no significant differences(P>0.05). Conclusion: Chemotherapy combined with metformin is more effective in the treatment of the patients with NSCLC and can reduce its adverse reactions, its mechanism may be related to the reduction of IGF-1 and mTOR levels in the peripheral blood of the patients.

Objective: To investigate the effect of dapagliflozin on the liver function of the patients with type 2 diabetes mellitus (T2DM) complicated with non-alcoholic fatty liver disease (NAFLD), and to analyze its improvement effect on liver injury of the T2DM patients. Methods: The clinical data of 68 T2DM patients complicated with NAFLDS who did not receive any treatment were retrospectively analyzed, including 30 patients in acarbose group and 38 patients in dapagliflozin group, and the patients in two groups received acarbose 150 mg·d^-1+ metformin 2 000 mg·d^-1 and dapagliflozin 10 mg·d^-1 + metformin 2 000 mg·d^-1 treatment for 24 weeks, respectively. The general data of the patients before and after treatment were collected.The fasting venous blood of the patients in two groups was collected and the serum biochemical indexes and liver function indexes were detected.The serum biochemical indexes included fasting blood glucose(FBG) and glycosylated hemoglobin (HbA1c),and the homeostasis model assessment 2-insulin resistance index(HMOA2-IR) was calculated.The liver function indexes included aspartic transaminase (AST), alanine aminotransferase (ALT), glutamyltranspeptidase (GGT), alkaline phosphatase (ALP), total bilirubin (TBIL),and direct bilirubin (DBIL);the non-alcoholic fatty liver disease fibrosis score (NAFLDFS) was calculated.The each detection index of the patients in two groups was compared and analyzed. Results: Compared with before treatment, the levels of FBG, HbA1c, AST, GGT, and ALP and HOMA2-IR in acarbose group after treatment were significantly decreased(P<0.01) and the levels of TBIL was significantly increased (P<0.01). Compared with before treatment, the levels of FBG, HbA1c, AST, ALT, GGT, and ALP and HOMA2-IR,NAFLDFS in dapagliflozin group were significantly decreased after treatment (P<0.01) and the levels of TBIL and DBIL were significantly increased (P<0.01). The levels of FBG, AST, ALT, GGT, and ALP and NAFLDF in dapagliflozin group after treatment were significantly decreased compared with acarbose group (P<0.01). Conclusion: Dagliflozin can improve the liver function of the patients with T2DM complicated with NAFLD, and its mechanism may be related to the effect of dapagliflozin decreasing the blood glucose and body weight.

Objective: To investigate the effects of Aominqing desensitizer and two laser (Er: YAG and Nd: YAG)treatments on the dentin surface morphology, hardness,and calcium(Ca) and phosphorus(P) contents and Ca/P ratio, to clarify the possible mechanisms, and to provide reference for its clinical application in the treatment of dentin sensitivity. Methods: The fresh third molars extracted because of impaction from the volunaeers aged 18-20 years old were collected.A total of 32 dentin slices of 2 mm were prepared; the samples were divided into blank control group, Aominqing group Er: YAG group and Nd: YAG group,and there were eight samples in each group.Except blank control group,the samples in other groups were used to establish the dentin hypersensitivity models by purifying the polluted layer with 17% EDTA. Each sample was cut by linear cutting machine from the middle symmetry into two semicircle dentin; the half was used to detect the dentin surface microhardness(SMH) values by microhardness meter,another half was used to observe the surface morphology and analyze the Ca and P contents with SEM and EDS was observed. Results: There were no significant differences in the SMH values of dentin samples between various groups (P>0.05). In blank control group, there was no smear layer on the dentin surface with wide tubules and smooth and clean surface;the dentin tubules of the samples in Aominqing group were partially closed, and obstructions were seen in the tubuless in Er: YAG group the dentin tubules were little or no open, most of them were partially or completely closed tubules, the surface had the melting changes in Nd: YAG group, the dentin tubules were partially or completely closed with few open tubules and the surface was molten.Compared with blank control group and Aominging group,the Ca and P contents in the samples in Er: YAG group and Nd: YAG were significantly increased; there was no statistically significant difference in the contents of Ca and P between and blank control group and Aclinsing group (P>0.05), and the difference between Er: YAG group and Nd: YAG group was not significant (P>0.05).The differences in the Ca/P ratios between various groups were not statistically significant (P>0.05). Conclusion: The application of Aominqing desensitization agent, Er: YAG laser and Nd: YAG laser can not cause the changes of dentin SMH and the Ca/P ratio, but the application of Er: YAG and Nd: YAG laser to dentin can significantly increase the contents of Ca and P and change the morphology, composition and structure of dentin surface.

Objective: To detect the thromboelastrography (TEG) parameters, mean platelet volume(MPV)/platelet count(PC) ratio and plasma fibrinogen(FIB) level in the patients with recurrent spontaneous abortionin(RSA) in non-pregnant state, and to evaluate their predictive values in the disease. Methods: A total of 206 patients with RSA history were selected as observation group and 92 healthy women who underwent prenatal examination during the same period were selected as control group. The TEG parameters, MPV, PC and FIB levels of the subjects in two groups were measured, and the predictive values of relevant indicators in RSA were evaluated. Results: Compared with control group, the setting time (K value) of TEG parameters of the patients in observation group was decreased (P<0.05), the setting angle(α angle) and maximum amplitide(MA value) were increased (P<0.05), and the MPV/PC ratio and FIB level were increased (P<0.05). There was no significant difference in the response time(R value) of TEG parameters of the subjects between two groups (P>0.05).The areas under receiver operating characteristic (AUC) curve of MA value, MPV/PC ratio and FIB level were 0.694, 0.673 and 0.735,respectively. The results showed that the three paramerters had good clinical predictive values in RSA. Conclusion: The K value, α angle, MA value, MPV/PC ratio and FIB level in the RSA patients have changed in the non-pregnant state, which is of great value in the early prediction of RSA.

Objective: To investigate the changes of the levels of amino-terminal pro-brain natriuretic peptide (NT-pro BNP) and lipoprotein a[Lp(a)], and to clarify the relationships between the severity of coronary artery lesions and the prognosis in the patients with acute myocardial infarction (AMI) after percutaneous coronary intervention (PCI). Methods: A total of 316 AMI patients underwent emergency PCI were selected. According to the number of stenosed coronary vessels, the patients were divided into single-vessel disease group(n=135), double-vessel disease group(n=99) and three-vessel disease group(n=82). According to the Gensini score, there were 79 cases in <38.63 group, 79 cases in 38.63-56.25 group, 79 cases in 56.26-83.00 group, and 79 cases in >83.00 group. The patients' general data, biochemical parameters, echocardiography results, and coronary angiography findings were recorded, and the major adverse cardiovascular events (MACE) were also recorded during a 12-month follow-up. The relationships between the serum NT-pro BNP and Lp(a) levels of the AMI patients in different lesion counts and Gensini scores, and the recent occurrence of MACE were analyzed. The receiver operating characteristic (ROC) curve was drawn to investigate the values of NT-pro BNP and Lp(a) levels on predicting the recent MACE in the patients with AMI. Results: The serum NT-pro BNP levels of the patients in three-vessel disease group and double-vessel disease group were higher than that in single-vessel disease group (P<0.01); the serum Lp(a) level of the patients in three-vessel disease group was higher than that in single-vessel disease group (P<0.05). According to Spearman correlation analysis, there were positive correlations between the serum NT-pro BNP level, Lp(a) level of the AMI patients and the number of coronary vessels(r=0.285, P<0.01;r=0.144, P=0.010); there were positive correlation between the serum NT-pro BNP level, Lp(a) level and Gensini score of coronary lesions (r=0.156, P=0.006;r=0.164, P=0.003). The serum NT-pro BNP and Lp(a) levels of the patients with MACE during follow-up were higher than those in the patients without MACE.The ROC curve showed that the area under the curve(AUC) of the levels of serum NT-pro BNP and Lp(a) was 0.747 (95%CI: 0.679-0.814). Conclusion: The serum NT-pro BNP and Lp(a) levels have the certain relationships with the severity of coronary artery lesions in the AMI patients after PCI. The simultaneous detection of serum NT-pro BNP and Lp(a) levels has certain predictive value for the recent occurrence of MACE in the AMI patients underwent emergency PCI.

Objective: To observe the preventive effect of low-dose aspirin (LDA) in the preeclampsia(PE) high-risk pregnant women, and to elucidate the feasibility and clinical value of LDA in preventing PE. Methods: A total of 112 PE high-risk pregnant women with 16-22 gestational weeks were selected and randomly divided into control group(n=58) and observation group(n=54). The patients in control group were given placebo treatment, the patients in observation group were given 75 mg·d^-1 LDA before going to bed until delivery.The incidence of PE, gestational age of onset, gestational age of delivery,neonatal birth weight, delivery mode, neonatal outcomes of the patients in two groups were observed.The patients in two groups were divided in to PE(+) (with PE) group and PE(-) (without PE) group according to whether PE occured or not. The coagulation function and the platelet parameters of the patients in two groups were detected; the levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α), thromboxane A2(TXA2),P-selectin in placenta tissue of the patients in two groups were detected by ELISA method. Results: Compared with control group,the incidence of PE and the incidence of premature delivery of the patients in observation group were significantly decreased(P<0.05);the gestational age of delivery of the patients in observation group were prolonged(P<0.05), the neonatal birth weight was increased(P<0.01),and the full-term birth rate was increased(P<0.01). Compared with the PE(+) and PE(-) patients in control group, the prothrombin time(PT) and activated partial thromboplastin time(APTT) of the PE(-) patients in observation group were increased(P<0.01),and the PT of the PE(+) patients in observation group was decreased (P<0.01);the levels of prothrombin activity(PTA),fibrinogen(FIB) and D-dimer(D-D) of the PE(+) and PE(-) patients in observation group were decreased. Compared with control group,the levels of IL-6,TNF-α, COX-2, TXA2 and P-selectin in placenta tissue of the patients in observation group were decreased significantly (P<0.01). Conclusion: LDA treatment within 16-22 weeks of gestation can effectively prevent the occurrence of PE in the PE high-risk pregnant women, its mechanism is related to improving the coagulation function and inhibiting the protein expressions of inflammatory factors in the PE high-risk pregnant women.

Objective: To explore the relationships between the follicle-stimulating hormone (FSH) level and the lipid profiles in the post-menopausal women, and to provide evidence for assessing the risk of dyslipidemia in the post-menopausal women. Methods: A total of 129 women with menopause for more than 1 year were selected as the subjects and divided into low FSH group(FSH<57.6 IU·L^-1) and high FSH group (FSH ≥ 57.6 IU·L^-1) according to the median of FSH. The data including height, weight, body mass index(BMI), age, menopausal age, duration of menopause, history of smoking, chronic diseases history of the subjects were collected; the serum total cholesterol(TC), triglyceride(TG), low density lipoprotein cholesterol(LDL-C), high density lipoprotein cholesterol(HDL-C), non-density lipoprotein cholesterol(non-HDL-C), estradiol(E2), FSH, fasting blood glucose, serum uric acid of the subjects were detected with the same method. Multivariate Logistic regression analysis was applied to analyze the relationship between the FSH level and dyslipidemia. Results: The average age of the post-menopausal women was (61.22±7.30) years, the age of menopause was (49.97±4.00) years, and the duration of menopause was (11.16±7.98) years. The proportion of post-menopausal women with dyslipidemia was 65.9%(85/129), and there was no significant difference between two groups(P>0.05).Compared with high FSH group, the TG level of the subjects in low FSH group was increased(P<0.01) and the HDL-C level was decreased(P<0.01). There were no significant differences in the TC, LDL-C and non-HDL-C levels between two groups. The Logistic regression analysis showed that after adjusting the E2 level, age, duration of menopause, hypertension history, diabetes history, fatty liver and the BMI grade, FSH level had a negative correlation with TG increase(≥ 2.3mmol·L^-1)(P<0.05). Each 10-unit increase in FSH was associated with a 29.5% lower risk of elevated TG (95%CI: 3.5%-48.5%). FSH was not significantly associated with the elevated TC(≥ 6.2mmol·L^-1), elevated LDL-C(≥ 4.1mmol·L^-1), decreased HDL-C(<1.0 mmol·L^-1) and elevated non-HDL-C. Conclusion: The FSH level is negatively associated with elevated TG inthe post-menopausal women, suggesting that low FSH appears to be a risk factor of dyslipidemia in the post-menopausal women.

Objective: To explore the effect of enhancing the rate of programmed intermittent epidural bolus(PIEB) on the labor analgesia and the dosage of ropivacaine supplement in the lying-in women,and to provide the basis for studing labor analgesia. Methods: One hundred and twenty-six women with a singleton pregnancy received labor analgesia with PIEB method and were randonly divided into low-rate group (n=60) and high-rate group (n=66). Epidural infusion was given the initial loading dose of 10 mL(0.09% ropivacaine+0.4 mg·L^-1 sufentanil), followed by 100 mL pulse injection pump(0.09% ropivacaine+0.4 mg·L^-1 sufentanil).Every 60 min, intermittent bolus of 10 mL was given; the patient were administered with the rates of 100 mL·L^-1(low-rate group) or 200 mL·L^-1 (high-rate group).The drug administration time of patient-controlled epidural analgesia (PCEA) was set as 5 mL,and the locking time was set as 30 min. The initial pain visual analog scale (VAS) score, duration of labor, delivery mode, supplementary amount and frequency of ropivacaine, first supplementary time of ropivacaine,a mount of PCEA pump, maternal satisfaction score, maximum sensory block level, as well as the incidence of adverse events,such as nausea and vomiting, hypotension, respiratory depression and fever of the lying-in women in the analgesia period were recorded. Results: The initial pain VAS scores, duration of labor, natural delivery rates, assisted vaginal delivery rates and cesarean section rates of the patients in two groups had no significant differences (P>0.05).There were no significant differences in the amount and frequency of supplementary, the first supplement time of ropivacaine,the amount of PCEA pump between two groups(P>0.05).The satisfaction scores of the lying-in women in two groups had no significantly difference(P<0.05).The highest analgesia level in two groups was T7-T8, and no adverse events,such as nausea and vomiting,hypotension, respiratory depression and fever, were observed in all the lying-in women. Conclusion: Compared with low-rate PIEB labor analgesia,the effect of labor analgesia, times of need for supplemental analgesia and the consumption of ropivacaine per hour are not improved by high-rate PIEB.

Objective: To analyze the reasons of rhabdomyolysis (RM) misdiagnosed with diabetetic ketoacidosis (DKA), to clarify the mechanisms of metabolic acidosis caused by RM and DKA,and to enhance the clinical understanding of RM and metabolic acidosis. Methods: The general situation, clinical manifestation, laboratory examination results of the patient who was admitted with DKA and was diagnosed as RM in our department, were collected. Based on the literatures, the causes and manifestations of RM, as well as the treatments and prognosis of the acute kidney injure (AKI) induced by RM, and the reasons of RM of the presented patient were analyzed; the similiarities and differences of the mechanisms and treatments of metabolic acidosis caused by AKI induced by RM and DKA were discussed. Results: The patient who was a 56 year-old female with hyperlycemia for 15 years, and fatigue, nausea and vomiting for 4 d was admitted. The physical examination results showed facial edema, dry tongue, poor skin elasticity, heart rate 110 min^-1, severe depressed edema of extremities, muscle strength Ⅳ level. The anxiliary examination results demonstrated that the levels of blood muscle enzymes, myoglobin, urea nitrogen and creatinine were increased; the blood gas analysis indicated metabolic acidosis, accompanying with electrolyte disturbance and abnormal blood routine. According to the history, symptoms and signs, as well as the laboratory test results, the patient was diagnosed as RM, AKI, metabolic acidosis combined with respriatory alkalosis, electrolyte disturbance, and so on.The clinical symptoms and signs of the patient were recovered, and the blood creatine kinase, myoglobin and renal fuction were significantly improved after the treatment of adequate volume replacement, alkalization and protection of vital organs; the patient had a good prognosis. Conclusion: The diabetic patients may suffer from metabolic acidosis due to various causes which should be paid attention to differential diagnosis. There are some differences of the mechanisms and treatments of metabolic acidosis caused by AKI and DKA. If given the treatment of adequate volume replacement, alkalization early and aggressively, the metabolic acidosis caused by RM combined with AKI can have an excellent prognosis.

Objective: To analyze the clinical characteristics,the typical pathological features and treatment methods of the patients with congenital hepatic fibrosis (CHF),and to improve the prognosis of the patients. Methods: The clinical data of a patient with CHF underwent liver transplantation for liver function failure were collected,and the relevant literatures were reviewed; the classification, clinical characteristics, diagnosis and treatment of CHF were analyzed. Results: The patient was a 39-year-old man and prensented with upper gastrointestinal bleeding. The results of gastroscope examination showed the severe varicose veins of the esophagus,which was marked by portal hypertension. The imaging examination after admission showed liver cirrhosis, splenomegaly and right renal cyst; the patient had mild liver function change, poor coagulation function, and liver function failure. After medical treatment, the condition was not improved, and the patient underwent allogeneic orthotopic liver transplantation. The postoperative liver pathology indicated CHF; all the laboratory indicators were normal after 1 month of transplantation. The patient recovered better 1 year after operation. Conclusion: CHF is characterized by liver fibrosis, portal hypertension and renal cystic lesions. Liver biopsy is the gold standard for its diagnosis, clinical treatment is mainly for the complications, and liver transplantation is the fundamental treatment.

Objective: To analyze the hematological changes of systemic lupus erythematosus(SLE) and the clinical characteristics of immune-related hypoglycemia,and to provide the basis for the diagnosis and treatment of SLE complicated with autoimmune hypoglycemia(AIH). Methods: The clinical data a patient with SLE complicated with AIH with pancytopenia as the first manifestation were collected and the relevant literatures were reviewed. Results: A 70-year-old man was admitted to hospital because of dizziness and fatigue,and suffered from more than 3 months,aggravated for 2 weeks. The physical examination results showed pale conjunctiva,moist rales over the both lower lung and there were no other obvious positive signs. The blood test showed pancytopenia and the fasting blood glucose 2.34 mmol·L^-1.The pathomorphology of tissue was observed by bone marrow puncture;rheumatism examinations,glucose metabolism indexes,insulin autoantibodies(IAA) and other assistant examinations were performed,and the patient received the related treatment.The patient had a history of photosensitivity. Admission examinations indicated multiple serous effusions,urinary protein >0.5g·24 h^-1,pancytopenia, abnormal antinuclear antibody(ANA) titer, pancreatic CT(-), and the patient was diagnosed as SLE complicated AIH finally.After treatment of prednisone,the symptoms of the patient were improved; the the whole blood count and fasting blood glucose responded well to the therapy of prednisone. After discharge from the hospital,the patient was treated with prednisone continuously and was required to regularly monitor the blood test and the blood glucose level. The patient's whole blood count was gradually increased and no hypoglycemia occurred. Conclusion: SLE with hematological changes as the first manifestation is easily misdiagnosed. And autoantibody-mediated glucose homeostasis should be considered when SLE is complicated with hypoglycemia.

Objective: To establish a new method for rapid detection of Coxsachie virus A16 (CA16) hand, foot and mouth disease pathogens based on fluorescence resonance energy transfer (FRET) technique, to evaluate the detection effect and to make the method to meet the requirements of large sample size detection during the outbreak of disease. Methods: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and bicinchoninic acid (BCA) protein assay were used to identify the purity of CA16 chicken yolk antibody(CA16-IgY) and the protein level. Indirect enzyme-linked immunosorbent assay (iELISA) was used to detect the titer and specificity of anti-CA16 IgY antibody. The size, morphology and characterization of gold nanoparticles (AuNPs) and their biological probes (IgY-AuNPs) were determined by UV-visible spectroscopy (UV-Vis), infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The CA16 detection system was constructed based on FRET technique. The sensitivity and specificity of the detection method and clinical sample detection were evaluated by optimizing the IgY-AuNPs concentration, sodium chloride (NaCl) dosage, fluorescence recovery time and other indicators. Results: The CA16-IgY had high purity, the titer was 1: 128 000,the average protein level was 12.15 mg·L^-1,and CA16-IgY had good specificity. The results of UV-Vis, FTIR and TEM of AuNPs and IgY-AuNPs showed that IgY was successfully labeled onto the surface of AuNPs, which suggested that IgY-AuNPs could specially recognize CA16 was successfully prepared by electrostatic self-assembly. The CA16 detection system was constructed based on FRET technology, after optimization of the detection system, the optimal dosage of IgY-AuNPs was determined to be 0.52×10^-3 g·L^-1, the optimal dosage of NaCl was 40 μL and the optimal fluorescence recovery time was 90 min. The standard curve of the established detection method was I_{525 nm}=15.452 IgC-9.746, R²=0.993 2, the detection limit was as 1×10⁴ PFU·mL^-1. Compared with qRT-PCR, the agreement rate reached 93.75%. Conclusion: A new rapid detection method for CA16 hand, foot and mouth disease pathogens is successfully established, which can be applied to laboratory and clinical tests.

Objective: To investigate the long-term expression of the piggyback(PB) transposon system expressing human interleukin-6(IL-6), interleukin-3(IL-3), interleukin-15(IL-15), stem cell factor(SCF) and granulocyte-macrophage cstimulating Factor(GM-CSF) genes in the immunodeficient mice, and to provide a simple,long-term and new method for improving the reconstruction of human immune cells in the humanized mouse models. Methods: The PB transposon plasmid (PB-GFP) containing the GFP gene was constructed, and the PB transposon plasmid (PB-5F) containing human IL-6, IL-3, IL-15, SCF, and GM-CSF genes was constructed.The 293T cells were divided into negative control group(non-transfection), positive control group(transfected with pLVTHM), transient transfection group transfected with PB-GFP plasmid and stable transfection group[transfected with PB-GFP plasmid together with transposase plasmid (super-PB)].The proportions of GFP⁺ cells in varions groups were measured by flow cytometry every three days after transfection. The NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ(NCG) mice were divided into transient transfection group and stable transfection group. The mice in transient transfection group were transfected with PB-GFP alone, and the mice in stable transfection group were transfected with PB-5F and super-PB. On the 1st, 4th, 5th and 9th days and the following every week after the transfection, the blood samples were collected, and the serum was separed;the levels of IL-6, IL-3, IL-15, SCF, and GM-CSF in serum of the mice in various groups were detected by ELISA. Results: At 30 d after transfection of PB-GFP, the percentage of GFP⁺ cells of the mice in stable transfection group (4.61%±0.42%) was significantly higher than those in positive control group (0.58%±0.05%) and transient transfection group (0.86%±0.10%) (P<0.05). At 30 d after transfection of PB-5F plasmid, the levels of serum IL-6, IL-15 and GM-CSF of the NCG mice in stable transfection group were significantly higher than those in transient transfection group (P<0.05); at 60 d after transfection, the levels of IL-6, IL-15 and GM-CSF in serum of the mice in stable transfection group were also found. Conclusion: The long-term expression of the PB (piggyBac) transposon system is verified in vitro, and the PB transposon system expressing human IL-6, IL-3, IL-15, SCF and GM-CSF genes is successfully constructed.An immunodeficient mouse model with long-term and stable expressions of human IL-6,IL-15,and GM-CSF is established.

Objective: To explore the development methods and preliminary clinical validation of thrombosis susceptibility gene chip, and to illustrate a rapid and high-throughput method for detecting the thrombosis susceptibility gene mutation. Methods: According to the published gene sequences of thrombotic susceptibility genes in GenBank, the reference probes and special probes were pointed on the glass slide. After ultraviolet crosslinking, thrombosis susceptibility gene chip was eatablished. The validity of gene chip was tested by positive reference (mutant gene and normal gene at each detection site) and negative reference (distilled water) as templates, thereby performing PCR reaction. The specificity and sensitivity of gene chip were detected by using the human genome DNA of target sequence proven by sequencing as templates, thereby performing PCR reaction. Meanwhile 150 healthy subjects and 24 thrombosis patients with family history of unexplained thrombotic disease from Jilin province, Henan province and Yunnan province were carried out the clinical verification of gene chip. The analysis index was the hybridization signal strength of the corresponding site. Results: The hybridization results of positive reference and negative reference as templates showed that the specific hybridization signals appeared at the corresponding sites, which indicated that the detection sites of gene chip are effective. The gene chip used to detect the selected mutation sites had specific hybridization signals, suggesting there were good specificity of gene chip. The sensitivity of gene chip was 50-100 mg·L^-1 by testing genomic DNA with stepwise dilution. Eight individuals with thrombosis susceptibility gene mutations were found in 150 healthy subjects. Twenty individuals with thrombosis susceptibility gene mutations were found in 24 thrombosis patients with family history of unexplained thrombotic disease. The statistcs results showed that the detection rate of thrombosis susceptibility gene mutations in the thrombosis patients with unexplained thrombotic disease family history was significantly higher than that of the healthy subjects (P<0.05). Conclusion: The developed thrombosis susceptibility gene chip has great specificity, sensitivity, and high detection rate of thrombosis susceptibility genes. It has potential values in the early diagnosis and susceptibility risk assessment of thrombotic diseases.