Objective: To transfer the pEgr1-TRAIL recombinant plasmid into human breast cancer MCF-7 cells and to combine with ionizing radiation,and to discuss the changes of related gene and protein expressions of death receptor pathway in breast cancer MCF-7 cells. Methods: The MCF-7 cells were divided into empty plasmid,pEgr1-TRAIL plasmid,4.0 Gy X-ray,empty plasmid+4.0 Gy X-ray, and pEgr1 TRAIL+4.0 Gy X-ray groups.Real-time PCR method was used to detect the expression levels of DR4,caspase-9,and caspase-6 mRNA in the MCF-7 cells in various groups;Western blotting method was used to detect the relative expression levels of DR4,caspase-9,and caspase-6 protein in the MCF-7 cells in various groups. Results: Compared with control group, the mRNA expression levels of DR4,caspase-9 and caspase-6 in MCF-7 cells in the other groups began to rise 4 h after 4.0 Gy X-ray irradiation(P<0.01),and they reached to the highest levels 8 h later,then reduced to the level of pre-irradiation 24 h later.The order of these mRNA expression levels from high to low in various groups was pEgr1-TRAIL+4.0 Gy X-ray group>empty plasmid+4.0 Gy X-ray group >pEgr1-TRAIL plasmid group >empty plasmid group >4.0 Gy X-ray group>control group;the expression levels of RD4,caspase-9,and caspase-6 mRNA in the MCF-7 cells in pEgr1-TRAIL+4.0 Gy X-ray group were higher than those in other groups (P<0.01).The relative expression levels of DR4,caspase-9 and caspase-6 proteins began to rise 6 h after irradiation, and reached the peak 12 h later; the relative expression levels of those protein 48 h later were still higher than those 6 h after irrdiation.The order of these protein expression levels from high to low in various group was pEgr1 TRAIL+4.0 Gy X-ray group >empty plasmid+4.0 Gy X-ray group >4.0 Gy X-ray group >pEgr1-TRAIL plasmid group >empty plasmid group >control group.The relative expression levels of DR4,caspase-9,and caspase-6 proteins in the MCF-7 cells in pEgr1-TRAIL+4.0 Gy X-ray group were higher than those in the other groups. Conclusion: pEgr1-TRAIL recombinant plasmid combined with radiation can kill and induce the apoptosis of MCF-7 cells,and its effect is better than recombinant plasmid or ionizing radiation used alone.

Objective: To construct the Epstein-Barr virus (EBV) latent membrane protein 2A(LMP2A) and gramulocyte-macrophase colony stimulating factor(GM-CSF) fusion gene (CSF2A) recombinant eukaryotic expression vector,and to discuss the effect of CSF2A fusion protein on the proliferation and apoptosis of EBV positive(EBV⁺) tumor cells. Methods: According to the principle of overlap extension,the LMP2A and GM-CSF gene fragments were restructured by connecting the coding (Gly4Ser) 3 polypeptide gene fragments of DNA.The fusion gene was connected with the pIRES2-eGFP eukaryotic expression vector by recombinant DNA technology,and the electrophoresis analysis of enzyme digestion and DNA sequencing methods were used to identify the recombinant plasmid.The pIRES2-CSF2A plasmid was established as experimental group,while the pIRES2-LMP2A plasmid and the pIRES2-eGFP plasmid were established as control groups.Then all the plasmids were transfected into the dendritic cells (DC),respectively.The expression levels of CSF2A gene and protein were tested by RT-PCR and Western blotting methods,separately,and the inhibitory rate of proliferation and apoptotic morphology of the EBV⁺ tumor cells in various groups were determined by MTT and Hochest methods. Results: The enzyme digestion experiment and sequence determination results showed that the CSF2A fusion gene was inserted into the pIRES2-eGFP plasmid,and the recombinant eukaryotic expression vector named pIRES2-CSF2A was successfully obtained.The CSF2A protein expression was found in DC after transfected with pIRES2-CSF2A.The MTT results prompted that the inhibitory rate of proliferation of the cells in pIRES2-CSF2A plasmid transfection group was obviously higher than those in control groups in a time-dependent manner.The Hochest test results showed the morphological changes of apoptosis and apoptotic body.The number of apoptotic cells in pIRES2-CSF2A group was increased significantly at 48 h after treatment. Conclusion: The recombinant eukaryotic expression vector pIRES2-CSF2A is effectively transfected into DC,and the fusion gene could significantly inhibit the proliferation of EBV⁺ CNE2 cells and also promote the apoptosis.

Objective: To discuss the occurrence of hypoxia-induced autophagy in the olfactory ensheathing cells (OECs) from olfactory mucosa in the newly-born SD rat models,and to evaluate the effect of autophagy on the cell proliferation. Methods: The OECs were obtained and cultivated by enzyme digestion and advanced Nash differential attachment,and the properties and purities of OECs were identified by immunofluorescent assay;the OECs cultured in hypoxia condition with 2% oxygen levels were used as hypoxia group,the cells cultured under the normal culture conditions were regarded as control group, and the OECs treated with autophagy inhibitor 3-MA and cultured under hypoxic conditions were regarded as 3-MA+hypoxia group.The morphology of cells in various groups was observed under phase contrast microscope; Western blotting method was used to measure the protein expression levels of hypoxia inducible factor-1(HIF-1α ),LC3Ⅰ/Ⅱ (autophagy marker protein) and Beclin-1 (autophagy marker gene) in the cells in control group,4 h hypoxia group,and 8 h hypoxia group;CCK-8 method was used to measure the influence of autophagy in cell proliferation in control group, 3-MA group,hypoxia group and 3-MA+hypoxia group, Results: After separated and purificated,the OECs in control group,hypoxia group and 3-MA+hypoxia group showed fusiform, typical bipolar,and tripolar cell morphology,and had no significant differences.In the cultivated OECs,the NGFRp 75 and GFAP were expressed positively through separation and purification,and the purity was 87%. Compared with control group,the expression level of HIF-1α in the OECs in hypoxia group was increased (P<0.05),and the expression levels of LC3I Ⅱ and Beclin-1 in the OECs in hypoxia group were increased (P<0.05).Compared with hypoxia group,the proliferation ability of OECs in 3-MA+hypoxia group was reduced. Conclusion: The autophagy induced by hypoxia can decrease the cell proliferation of OECs from olfactory mucosa in the newly-born SD rats.

Objective: To observe the effect of rimonabant on the susceptibility of epilepsy in the rats after head injury,and to explore whether rimonabant can block epilepsy in the rats after head injury. Methods: A total of 105 rats were divided into model-management group (n=45),model group (n=45), and blank control group (n=15).The rats in model-management group and modeling group were used to establish the traumatic head injury models by fluid percussion injury (FPI) method,and the rats were intraperitoneally administered with rimonabant and saline 2 min after FPI,respectively.The rats in model and model-management groups were intraperitoneally injected with pentylenetetrazole at 24 h,1 week, and 6 weeks after FPI.The rats in blank control group were also intraperitoneally injected with pentylenetetrazole.The latency of epilepsy and the number of generalized tonic clonic seizures(GTCS) of the rats in each group were observed and compared. Results: In model group,the latency of epilepsy of rats at 6 weeks after PFI was obviously shorter than those at 24 h after FPI(F=12.93,P=0.023) and 1 week after FPI(F=11.80,P=0.016).However,in model-management group,the latency of epilepsy of the rats at 24 h after PFI was shorter than those at 1 week after FPI(F=14.69,P=0.024) and 6 weeks after FPI(F=11.69,P=0.024).The latency of epilepsy of the rats in blank control group was longer than those at 6 weeks after FPI in model group(F=14.74,P=0.03) and at 24 h after FPI in model-management group(F=18.33,P=0.007).The latency of rats in model group at 24 h after FPI was longer than that at 24 h after FPI in model-management group(t=2.97,P=0.009);the latency at 6 weeks after FPI in model group was shorter than that at 6 weeks after FPI in model-management group (t=2.31,P=0.033). The number of rats with GTCS in model-management group was decreased at 6 weeks after FPI than that in model group(χ²=6.67,P=0.033). Conclusion: The rimonabant can increase the susceptibility of epilepsy in the acute stage of head injury and plays an opposite role in the chronic stage.

Objective: To identify the basic chemical compositions of panax ginseng glycoprotein,and to discuss its influence in the scopolamine-induced study and memory impairment in the mice. Methods: Hollow fiber ultrafiltration device was used to separate the ginseng aqueous extract which saponin had been removed.The content of total sugar was determined by phenol sulfuric acid method;the content of protein was determined by Lowry method;the purity and relative molecular weight distribution were determined by HPLC;the sugar compositions were analyzed after the preparation of PMP derivatives.The mouse model with acquired memory disorder was established through injection of scopolamine.A total of 60 mice were randomly divided into control group(normol mice),model group(intraperitoneal administration of scopolamine hydrobromide 2.5 mg·kg^-1 after training),positive drug group(intragastric administration of piracetam 9 mg·kg^-1 for 30 d before training),and low,middle and high doses of Panax ginseng glycoprotein groups (intragastric administration of glycoprotein 7.5,75.0,225.0 mg·kg^-1 respectively for 30 d before training),and there were 10 rats in each group.The escape latency and the number of crossing the platform of the mice were determined by Morris water maze and step-down test. Results: The total sugar mass fraction of Panax ginseng glycoprotein was 58.41% which was consisted of 7 kinds of sugars.The mass fraction of protein was 19.93% which was consisted of 17 kinds of amino acids.The relative molecular weights of Panax ginseng glycoprotein were 200-50 000.The Morris water maze test results showed that from the 3rd day,the average escape latency of mice in high dose of Panax ginseng glycoprotein group was significantly shortened compared with model group (P<0.05),and the number of crossing the platform in high dose of Panax ginseng glycoprotein group was significantly more than that in model group (P<0.01).The relative position test results showed that the time of spenting in the platform quadrant s of the mice within 60 s in high dose of Panax ginseng glycoprotein group was significantly prolonged compared with model group (P<0.05);from the 2 day,the average escape latency in different doses of Panax ginseng glycoprotein groups was significantly shortened compared with model group (P<0.05).The step-down test results showed that the mice in different doses of Panax ginseng glycoprotein groups made significantly less mistakes within 3 min compared with model group(P<0.05);compared with model group,the latent period of landing on the platform of the mice in middle dose of Panax ginseng glycoprotein group was significantly increased(P<0.05). Conclusion: Panax ginseng glycoprotein could improve the memory ability of mice.

Objective: To investigate the effect of celecoxib combined with taxol on the apoptosis of breast cancer MCF-7 cells,and to explore the possible mechanisms of the synergistic effect of Cox-2 inhibitors combined with chemotherapy drugs. Methods: The MCF-7 cells were divided into blank control group(treated with normal medium),celecoxib group(treated with different concentrations of celecoxib),taxol group(treated with different concentrations of taxol),and celecoxib combined with taxol group(combination group,treated with celecoxib combined with taxol).Hoechst 33258 staining was used to observe the morphology of apoptotic cells;MTT assay was adopted to detect the survival rate of MCF-7 cells;flow cytometry with Annexin Ⅴ FITC/PI double staining was used to detect the distribution of cell cycle and the apoptotic rate of MCF-7 cells;Western blotting method was taken to detect the protein expressions of b-cell lymphoma-2(bcl-2),extracellular regulated protein kinase2(ERK2) and phosphorylase-extracellular regulated protein kinase(p-ERK) in the MCF-7 cells. Results: The Hoechst 33258 staining results showed that compared with blank control group,the number of apoptotic cells in both celecoxib group and taxol group were increased,and the number of apoptotic cells in combination group was the highest,and the number of apoptotic cells was increased with the increasing of celecoxib concentration.The MTT assay results showed that the survival rates of MCF-7 cells in celecoxib group and taxol group were decreased with the prolongation of treatment time;during the same period,the MCF-7 cells treated with the combination of 50 mg·L^-1 celecoxib and 30 μg·L^-1 taxol had a much lower survival rate compared with celecoxib or taxol used alone(P<0.05).The flow cytometry results showed that the MCF-7 cells in celecoxib group were arrested at G₀/G₁ phase,and the MCF-7 cells in taxol group were arrested at G₂/M phase;compared with blank control group,the distribution of cell cycle in combination group changed dramatically,such as the ratio of MCF-7 cells arrested at G₀/G₁ phase and G₂/M phase were increased,and the ratio of MCF-7 cells arrested at in S phase was decreased.The Annexin Ⅴ FITC/PI double staining results showed that 6 h after drug treatment,the apoptotic rates of MCF-7 cells in celecoxib group and taxol group were higher than that in blank control group(P<0.05),and the apototic rate of cells in combination group was the highest(P<0.05).The Western blotting results showed that compared with blank control group,the expression levels of bcl-2 and p-ERK proteins in MCF-7 cells in celecoxib group, taxol group and combination group were all decreased,especially in combination group;but there were no significant differences in the ERK2 protein expression levels between various groups(P>0.05). Conclusion: Celecoxib and taxol show a synergistic effect on inducing the apoptosis of MCF-7 cells and its mechanism may be related to the down-regulation of the expressions of bcl-2 and p-ERK proteins.

Objective: To construct interferon regulatory factor 5(IRF5) gene lentiviral vector LV11-cmv-neo-IRF5 and transfect it into macrophages RAW264.7, and to observe its expression in RAW264.7 cells. Methods: The target gene IRF5 gragment was fished by PCR and cloned into linearized lentiviral vector LV11 by using DNA recombinant technique. The recombinant vector was identified by PCR, double enzyme digestion,and sequencing and the 293T cells were transfected with the lipofectin reagent for lentiviral particles packaged with expression and packing plasmids. The supernatant of lentiviral was collected and infected into the macrophages RAW264.7. After screening the cells, which were not infected with G418, the positive infected cells(RAW264.7-IRF5)were obtained (experiment group). Meanwhile, the RAW264.7-NC cells was regarded as controls(control group).The expression levels of IRF5 mRNA and protein in RAW264.7 cells in two groups were examined by RT-qPCR and Western blotting methods. Results: The PCR and enzyme digestion analysis results confirmed that the lentiviral vector LV11-cmv-neo-IRF5 was successfully constructed. The RAW264.7 cells were infected with virus supernatant and the positive cells were obtained by G418 screening.The RT-qPCR results showed that compared with control group, the expression level of IRF5 mRNA in the RAW264.7 cells in experiment group was increased (P<0.01);the Western blotting results showed that compared with control group,the IRF5 protein epression level in the RAW264.7 cells in experiment group was increased. Conclusion: The lentiviral vector LV11-cmv-neo-IRF5 containing IRF5 gene is successfully constructed,and the IRF5 gene can express stably in the macrophages RAW264.7.

Objective: To investigate the effects of erlotinib on the inflammatory response of macrophages and acute lung injury(ALI ) induced by lipopolysaccharides(LPS)in the mice,and to reveal their mechanisms. Methods: The murine bone marrow derived macrophages were randomly divided into control group,erlotinib group,LPS group, and LPS+erlotinib group. The expression levels of tumor necrosis factor α(TNF-α) in the macrophages in various groups were detected by ELISA method and the phosphorylation levels of p38 and ERK1/2 were tested by Western blotting method. A total of 16 male mice were randomly divided into control group(saline),erlotinib group(45 mg·kg^-1 erlotinib for 3 d+saline),LPS group(saline+5 mg·kg^-1 LPS),LPS+erlotinib group(45 mg·kg^-1erlotinib+5 mg·kg^-1 LPS). The expression levels of TNF-α in the macrophages in serum was detected by ELISA method and the phosphorylation levels of p38 and ERK1/2 were tested by Western blotting method;the pathomorphology of lung tissue of the mice in various groups was observed by HE staining. Results: Compared with control group, the expression levels of TNF-α in the macrophages and serum of the mice,and the phosphorylation levels of ERK1/2 and p38 in the macrophages and lung tissue of the mice in erlotinib group had no significant difference(P>0.05),and the pathomorphology of lung tissue of the mice had no changes.Compared with erlotinib group,the expression levels of TNF-α in the macrophoages and serum of the mice and the phosphorylation levels of ERK1/2 and p38 in the macrophages and lung tissue of mice in LPS group were significantly increased(P<0.05),and the pathomorphology of LPS-induced ALI could be found.Compared with LPS group,the expression levels of TNF-α in the macrophages and serum of the mice and the phosphorylation levels of ERK1/2 and p38 in the macrophages and lung tissue of the mice in LPS+erlotinib group were decreased(P<0.05),and the pathomorphology of ALI was improved. Conclusion: Erlotinib can reduce the systemic inflammatory response of the ALI mice by inhibiting the phosphorylation levels of p38 and ERK1/2 of macrophages and the expression level of TNF-α to protect the ALI to a certain degree.

Objective: To explore the effects of interleukin-17 (IL-17) on the expression levels of matrix metalloproteinase-9 (MMP-9) in the osteoclasts, and to provide the references for the study on the effect of IL-17 on the orthodontic force-induced root resorption. Methods: The mouse monocytes/macrophages RAW264.7 were cultured and divided into experiment group and control group; the culture medium in experiment group were added with the gradient concentrations of 0.1, 1.0, 10.0, and 100.0 μg·L^-1 IL-17, and set as 0.1,1.0,10.0, and 100.0 μg·L^-1 IL-17 groups,and the culture medium in control group was IL-17-free.The RAW264.7 cells in various groups were induced to form osteoclasts and identified by TRAP staining on the 5th day after induction. ELISA method was used to detect the expression levels of MMP-9 in the culture medium of RAW264.7 cells in various groups when the osteoclasts were induced for 5 d; RT-PCR method was used to detect the expression levels of MMP-9 mRNA in the culture medium of RAW264.7 cells in various groups when the osteoclasts were induced for 5 d. Results: The TRAP staining results showed TRAP staining positive cells were found, with large volume and large number of nucleus (nucleus≥3) on the 5th day after induction. The ELISA and RT-PCR results showed the expression levels of MMP-9 and MMP-9 mRNA in the culture medium of RAW264.7 cells in 1.0,10.0 and 100.0 μg·L^-1IL-17 groups were increased compared with control group(P<0.05); the expression levels of MMP-9 and MMP-9 mRNA in the culture medium of RAW264.7 cells in 10.0 and 100.0 μg·L^-1 IL-17 groups were increased compared with 1.0 μg·L^-1 IL-17 group(P<0.05); the expression levels of MMP-9 and MMP-9 mRNA in the culture medium of RAW264.7 cells in 100.0 μg·L^-1 IL-17 group were increased compared with 10.0 μg·L^-1 IL-17 group(P<0.05). Conclusion: IL-17 can promot the expressions of MMP-9 in the osteoclasts, and the promoting effect is enhanced with the increasing of IL-17 concentration.

Objective: To explore the inhibitory effect of lidamycin (LDM) on the cellproliferation and apoptosis induction effect in the human chronic myeloid leukemia (CML) K562 cells,and to discuss the mechanisms. Methods: The K562 cells at logarithmic growth phase were selected and treated with different concentrations (0.01,0.10 and 1.00 nmol·L^-1) of LDM for 48 h and they were used as 0.01,0.10,and 1.00 nmol·L^-1 LDM groups,and the normal cells without treatment were regarded as control group.Trypan blue staining was used to detect the inhibitory rates of growth of the K562 cells in various groups;the cell viability was determined by MTT assay;the apoptotic morphology of cells was investigated by acridine orange/ethidium bromide (AO/EB) staining;the apoptotic rate of cells was detected by Annexin Ⅴ-FITC/PI double staining and flow cytometry;the relative expression levels of caspase-3 and caspase-8 in K562 cells were measured by Western blotting analysis. Results: The Trypan blue staining results showed that LDM could inhibit the proliferation of K562 cells and the inhibitory rates of growth of the K562 cells in different concentrations of LDM groups were higher than that in control group (P<0.05).The MTT results showed that the cell viability was decreased with the increasing of LDM concentration and the 50% inhibitory concentration (IC₅₀) value was (0.1 ± 3.2) nmol·L^-1.The AO/EB double staining results showed that LDM could induce the apoptosis of K562 cells under fluorescence microscope (the cytoplasm was in buds and the nucleus were broken into points).The flow cytometry results showed that the apoptotic rates of cells in different concentrations of LDM groups were higher than that in control group (P<0.05).The Western blotting analysis results showed that the relative expression levels of caspase-8 and caspase-3 in the K562 cells in 0.10 nmol·L^-1 and 1.00 nmol·L^-1 LDM groups were higher than those in control group (P<0.01);the relative expression levels of caspase-8 and caspase-3 in the K562 cells in 0.01 nmol·L^-1 LDM group had no significant differences compared with control group(P＞0.05). Conclusion: LDM could inhibit the proliferation of K562 cells.Moreover,low concentration of LDM may induce the apoptosis by up-regulating the expressions of caspase-8 and caspase-3.

Objective: To study the effects of 5- hydroxytryptamine 2C(5-HT_2C) receptors in the lateral habenular nucleus(LHb) on anxiety behaviors in the rats with Parkinson's disease(PD),and to clarify the neural mechanisms of PD associated anxiety behaviors. Methods: A total of 126 rats were randomly divided into sham operation group (n= 63) and substantia nigrapar compacta(SNc) lesion group(n=63).The anxiety behaviors of rats in two groups such as the percentges of the time spending in center region were observed by the open field test,and the percentages of number of open arm entries and time of open arm staying were observed by elevated plus maze test;the monoamine levels in limbic related brain regions were assessed by high performance liquid chromatography with electrochemical detection;the cannula position and the degeneration of dopamine (DA) neurons after SNc lesion were confirmed by histological and immunohistochemical staining. Results: Compared with sham operation group, the percentage of time spending in center region (F_1,72 = 36.678,P<0.001),the percentage of number of open arm entries (F_1,72 = 59.077,P<0.001) and the percentage of time of open arm staying (F_1,72 = 84.245,P<0.001) of the rats in SNc lesion group were decreased.The DA levels in medial prefrontal cortex (mPFC) (P<0.01),amygdala (P<0.01),and hippocampus (P<0.05) of the rats in SNc lesion group were significantly decreased compared with sham operation group. Compared with the rats treated with vehicle in the same group, the intra-injection of 5-HT_2C receptors agonist Ro60-0175 could significantly decrease the percentages of time spending in center region(P<0.01 or P<0.01),the percentages of number of open arm entries (P<0.05 or P<0.01),and the percentages of time of open staying(P<0.05 or P<0.01) and the monoamine levels in mPFC,amygdala and hippocampus of the rats.Three monoamine levels in mPFC of the rats in two groups were decreased significantly (P<0.05 or P<0.01);in amygdala,the DA and NA levels of rats in two groups were decreased significantly (P<0.05 or P<0.01);in hippocampus,all the monoamine levels except for the NA levels of the rats in sham operation group were significantly reduced(P<0.05 or P<0.01);the behavioral test results showed that the cannula position of rats was located at 1 mm above the LHb.The immunohistochemical staining results showed that the DA neurons of the injected side of the rats in SNc lesion group were missing,and the number of DA neurons in ventral tegmetal area was lower than that in unSNc area(P<0.001). Conclusion: The anxiety-like behaviors can be induced or enhanced after activation of LHb 5-HT_2C receptors,and their mechanisms may involve the changes of monoamine levels in the related brain regions.

Objective: To design and construct the expression vectors of α-catulin-shRNA,and to establish the TSCCA cell model with silencing α-catulin gene of tongue squamous cell carcinoma (TSCC) and observe its silencing efficiency,and to provide foundation for gene treatment of TSCC. Methods: The TSCCA cells were divided into transfection groups (transfected with sh-catu1,sh-catu2,sh-catu3, and sh-catu4),negative control group (transfected with sh-nc),and blank control group(untreated).According to the mRNA sequence of α-catulin obtained from GenBank, the expressing vectors of α-catulin-shRNA plasmids were designed and compounded.After the vectors were identified by DNA sequencing,the expressing vectors were transfected into the TSCCA cells cultured in vitro after mediated with liposome.The expression of green fluorescence protein (GFP) in TSCCA cells was observed by fluorescence microscope;the relative expression levels and silencing efficiency of α-catulin mRNA and protein were detected by RT-PCR and Western blotting methods. Results: The DNA sequencing results showed the plasmid expression vectors containing shRNA against α-catulin gene (sh-catu1,sh-catu2,sh-catu3,and sh-catu4) were constructed correctly.After transfection,the expressing vectors were expressed successfully in the TSCCA cells.The RT-PCR results showed that compared with blank control group,the relative expression levels of α-catulin mRNA in TSCCA cells in ttansfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05).Compared with negative control group,the relative expression levels of α-catulin mRNA in TSCCA cells in transfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05).The Western blotting results showed that compared with blank control group,the relative expression levels of α-catulin protein in TSCCA cells in transfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05).Compared with negative control group,the relative expression levels of α-catulin protein in TSCCA cells in transfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05). Conclusion: The recombinant plasmid vectors are constructed successfully,which could silence the α-catulin expression in TSCCA cells.This study will provide a new cell model for further study of the role of α-catulin in the pathogenesis of TSCC.

Objective: To explore the effects of silencing expression of α-catulin gene on the proliferation and apoptosis of human tongue squamous cell carcinoma(TSCC) TSCCA cells,and to clarify its role in the biological behavior of TSCCA cells. Methods: The TSCCA cells at logarithmic growth phase were selected and divided into α-catulin-siRNA transfection group,empty plasmid transfection group, and blank control group.The relative expression levels of α-catulin mRNA in the TSCCA cells in various groups were tested by RT-PCR method;the relative expression levels of α-catulin protein in TSCCA cells in various groups were detecetd by Western blotting method;the inhibitory rate of proliferation of TSCCA cells was checked by MTT method;The apoptotic rates of TSCCA cells were measured by flow cytometry(FCM). Results: The RT-PCR results showed that the relative expression level of α-catulin mRNA in the TSCCA cells in α-catulin-siRNA transfection group was lower than those in empty plasmid transfection group and blank control group(P<0.01); the relative expression levels of α-catulin mRNA were decreased gradually with the prolongation of time,and the relative expression level of α-catulin mRNA at 72 h was lower than those at 24 and 48 h(P<0.01).The Western blotting results revealed that compared with empty plasmid transfection group and blank control group,the relative expression level of α-catulin protein in the TSCCA cells in α-catulin-siRNA transfection group was decreased(P<0.05).The MTT results demonstrated that the inhibitory rate of growth of TSCCA cells in α-catulin-siRNA transfection group was higher than those in empty plasmid transfection group and blank control group(P<0.001).The FCM results exhibited that the apoptotic rate of TSCCA cells in α-catulin-siRNA transfection group was increased compared with empty plasmid transfection group and blank control group(P<0.001). Conclusion: Silencing the expression of α-catulin RNA specifically could suppress the growth of TSCCA cells and promote the apoptosis of TSCCA cells.

Objective: To investigate the effects of low molecular weight chitosan (LMCTS) on the expression of chcoride channel 3(CIC-3) in cardiomyocytes of the rats with hypoxia-reoxygenation(H/R)injury,and to probe the protective effect of LMCTS on the myocardial H/R injury and its correlation with the expression of ClC-3. Methods: The cardiomyocytes of rats were cultured and divided into blank control group,model group,and 200,400 and 600 mg·L^-1 LMCTS groups.The cells in blank control group were cultured regularly without any treatment;and the cells in other groups were treated under hypobaric and hypoxic condition for 1 h,then they got reoxygenation at 37℃ for 24 h to establish the cardiomyocyte H/R injury models.The cells in model group were cultured regularly without drugs and the cells in three different concentrations of LMCTS groups were given 200,400 and 600 mg·L^-1 LMCTS before establishing the H/R injury models.The ClC-3 mRNA expression level in cardiomyocytes of the rats was detected by RT-PCR method;the relative expression level ClC-3 protein in cardiomyocytes of the rats was detected by Western blotting method and immunofluorescence method. Results: The Western blotting and RT-PCR results indicated that the expression levels of ClC-3 mRNA and protein in cardiomyocytes of the rats in model group were higher than those in blank control group (P<0.01);but the expression levels in different concentrations of LMCTS groups were lower than those in model group(P<0.05),especially in 600 mg·L^-1 LMCTS group.The immunofluorescence experiment results showed that compared with blank control group,the fluorescence intensities in cardiomyocytes of the rats in model group were increased,but the fluorescence intensities in different concentrations of LMCTS groups were lower than that in model group. Conclusion: The ClC-3 expression in cardiomyocytes of the rats associates with the cardiomyocyte H/R injuriy.LMCTS could decrease the ClC-3 expression level and protect the cardiomyocytes away from H/R injury by the down-regulation of ClC-3 expression level.

Objective: To observe the effect of QingNaoZhiTong JiaoNang(QNZTJN) on the expressions of inflammatory cytokines in midbrain tissue and plasma of nitroglycerin(NGT)-induced migraine rats and its significance. Methods: Atotal of 90 male Wistar rats were randomly divided into control group,model group,low dose(0.35 g·kg^-1),middle dose(0.70 g·kg^-1),high dose(1.40 g·kg^-1) of QNZTJN groups and positive drug (Zhengtian Wan,1.8 g·kg^-1) control group(n=15).After treated with drugs for 7 d,the migraine models were induced by injecting of NTG into the rats;the forelimb scratching frequency of the rats during 180 min of NTG injection.The midbrain tissue and plasma were obtained 4 h later.The expression levels of nuckear factor kappa B(NF-κB) and cyclooxygenase 2(COX-2) in brain tissue of the rats were detected by immunohistochemistry method and Western blotting method;the levels of prostaglandin E₂(PGE₂),interleukin-1 beta(IL-1β), and tumor necrosis factor-α(TNF-α) in the midbrain tissue and plasma of the rats were detected by enzyme immunosorbent assay. Results: Compared with control group,the forelimb scratching frequency of the rats in model group was significantly increased(P<0.01),the levels of NF-κB and COX-2 in midbrain tissue were significantly increased(P<0.01),and the levels of PGE,IL-1β and TNF-a were increased in brain tissue and plasma(P<0.05 or P<0.01).Compared with model group,the forelimb scratching frequencies of the rats in middle and high doses of QNZTJN groups and positive drug control group were significantly decreased(P<0.05 or P<0.01),the levels of NF-κB and COX-2 in brain tissue were also significantly decreased(P<0.05 or P<0.01).The levels of PGE2,IL-1β and TNF-a in plasma were decreased (P<0.05 or P<0.01). Conclusion: QNZTJN can relieve the migraine attacks and prevent migraine partly and its mechanism may be related to regulating the neurogenic inflammation.

Objective: To detect the association between carcinoma cell oncogene epidermal growth factor receptor (EGFR) with L858R mutation and the expression levels of interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) in the carcinoma cells of lung adenocarcinoma patients with malignant metastasis and their significances. Methods: The vetor contained EGFRL858R full gene was transfect into the EGFR wild type lung adenocarcinoma H460 cell line(EGFRL858R-H460 group), the empty vector was transfected into the H460 cell line as control group(Vec-H460 group),and the expression levels of IL-6 and VEGF mRNA in the cells in two groups were detected by QRT-PCR method.The IL-6 siRNA was transfected into the lung adenocarcinoma H1975 cell line with EGFRL858R mutation( IL-6 siH1975 group),the empty vector was also transfected into the H1975 cell line as control group(Vec-H1975 group);the expression levels of IL-6 and VEGF mRNA in the cells in two groups were detected by QRT-PCR method,and the expression level of VEGF protein was detected by Western blotting method. Results: Compared with Vec-H460 group,the expression levels of IL-6 and VEGF mRNA in the cells in EGFRL858R-H460 group were increased (P<0.01); compared with Vec-H1975 group, the expression levels of IL-6 and VEGF mRNA in the cells in IL-6 siH1975 group were decreased (P<0.01),and the expression level of VEGF protein in the cells in IL-6 siH1975 group were decreased. Conclusion: The EGFRL858R mutation can elevate the expression levels of IL-6 and VEGF mRNA in lung adenocarcinoma cell lines,and the increasing expression of IL-6 mRNA may be responsible for the increasing expression of VEGF mRNA.These changes may be the evidences to prove that the EGFRL858R mutation is involved in the malignant transfer process in lung adenocarcinoma.

Objective: To establish the drug-resistant cell line CAL-27/DDP of human tongue squamous cell carcinoma,and to discuss the mechanism of cisplatin resistance. Methods: The CAL-27/DDP cells were induced by increasing the concentrations of cisplatin in vitro. Then the cells were divided into CAL-27 group and CAL-27/DDP group.The cells in CAL-27 group were teated with common nutrient solution and the cells in CAL-27/DDP group were treated with nutrient solution containing different concentrations of cisplatin. The resistance cell line CAL-27/DDP with good growth status was formed 12 months after induction.The morphology of cells was observed by inverted microscope;the half maximal inhibitory concentration(IC₅₀) value and drug resistance index(RI) of cisplatin in the CAL-27 and CAL-27/DDP cells were detected by MTT assay;the cell cycle distribution of CAL-27 and CAL-27/DDP was detected by flow cytometry;the relative expression level of MDR1 in the cells was detected by reverse transcriptase polymerase chain reaction (RT-PCR) method;the relative expression level of P-glycoprotein(P-gp) in the cells was detected by Western blotting method. Results: The cells in CAL-27/DDP group could stably grow in the ordinary medium in a period and maintained its morphology and resistance status.Compared with CAL-27 group,the cells in CAL-27/DDP group showed particles and "cavity" samples,plenty of plasma,and increased doubling time (P<0.01);the RI was 18.09±0.30.Compared with CAL-27 group,the ratios of cells at G₁ phase and G₂ phase in CAL-27/DDP group were decreased,and the ratio of cells at S phase and the relative expression level of MDR1 mRNA in the CAL-27/DDP cells were increased significantly(P<0.01),and the relative expression level of P-gp was increased (P<0.01). Conclusion: The drug-resistant cell line CAL-27/DDP of human tongue squamous cell carcinoma is established sucessfully and the drug-resistant mechanismmay be related to the up-regulation of MDR1 and P-gp expression levels in CAL-27/DDP cells.

Objective: To discuss the bonding strengths of commonly used three kinds of orthodontic adhesives under the dry and artificial saliva conditions,and to evaluate their bonding properties and provide the reference for clinical application. Methods: A total of 60 premolars removed for the orthodontic treatment were collected and randomly divided into 3M light curing adhesive group,3M chemical curing adhesive group, and Hangzhou West Lake enamel adhesive group,and there were 20 premolars in each group;then the each group was divided into two sub-groups with 10 premolars.A1 group was dry condition + metal brackets + 3M light curing adhesive,and A2 group was artificial saliva condition + metal brackets + 3M light curing adhesive; B1 group was dry condition + metal brackets + 3M chemical curing adhesive,and B2 group was artificial saliva condition+metal brackets + 3M chemical curing adhesive;C1 group was dry condition + metal + Hangzhou West Lake enamel adhesive,and C2 group was artificial saliva condition + metal brackets+ Hangzhou West Lake enamel adhesive.The electronic universal mechanical testing machine was uesd to test the bond strengths of orthodontic adhesives in various groups under the dry or artificial saliva conditions. The microscope was used to observe the adhesive remnant index(ARI) of orthodontic adhesives on the surface of the teeth, which was bonded with metal brackets;the scanning electron microscope was used to observe the surface morphology of the teeth detached from metal brackets. Results: Under both dry and artificial saliva conditions,the bonding strength of orthodontic adhesives in 3M light curing adhesive group was higher than those in 3M chemical curing adhesive and Hangzhou West Lake enamel adhesive groups(P<0.05); the bonding strength of orthodontic adhesives in 3M light curing adhesive group under the dry condition was higher than that under artificial saliva condition. Under the saliva condition, the bonding strengths of orthodontic adhesives in varous groups had no significant differences(P>0.05);the ARI of premolars in 3M light curing adhesive group was lower than those in other two groups(P<0.05).The testing results of scanning electron microscope showed that compared with the other two groups,the surface of premolars in 3M light curing adhesive group was more regular and flat without obvious cracks and enamel stripping. Conclusion: Under different conditions,the bonding strengths of three kinds of orthodontic adhesives can meet the requirements of clinical orthodontics,and the bonding properties of orthodontic adhesives of 3M light curing adhesive is better than the other two kinds of orthodontic adhesives.

Objective: To compare the corrosion resistance abilities between diamond-like carbon (DLC)coated nickel-titanium arch(NiTi) wires and uncoated NiTi wires in the artificial saliva with different pH values and fluoride(F^-)concentrations, and to provide the basis for the application of DLC in the orthodontics treatment. Methods: The DLC coated NiTi wires were prepared by radio-frequency magnetron sputtering technique. Raman spectrum was used to characterizate the NiTi wires. The DLC coated wires were taken as experiment group, and the uncoated NiTi wires were taken as control group, and the NiTi wires in two groups were exposed to four kinds of artificial saliva as four groups, pH7＋0%F^- group: pH 7+0.2% F^- group,pH7+0.5% F^- group,and pH5+0.5% F^- group.The potentiodynamic polarization (Tafel)curves of NiTi wires in two groups were measured by the classic three electrode system of electrochemical workstation,and the corrosion potential (Ecorr) and the corrosion current density (Icorr)of the NiTi wires in two groups were detected; scanning electron microscope(SEM) was used to observe the morphology of the NiTi wires in two groups before and after corrosion with artificial saliva. Results: The Raman spectra results showed the typical D peak and G peak,which indicated that the NiTi wires had been coated by DLC. The electrochemical corrosion test showed that compared with pH7+0% F^- group,the Ecorr of DLC coated NiTi wires in pH7+0.2% F^- group was decreased(P<0.05)and the Icorr was increased(P<0.05);the Ecorr of uncoated NiTi wires was decreased(P<0.05),and the Icorr was increased,but there was no significant difference(P>0.05).Compared with pH7+0.5% F^- group, the Ecorr of DLC coated NiTi wires and uncoated NiTi wires in pH5+0.5% F^-group were decreased(P<0.05)and the Icorr were increased(P<0.05). Meanwhile,in the same group of artificial saliva,the Ecorr of NiTi wires in expiment group was apparently higher than that in control group(P<0.05)and the Icorr was lower than that in control group(P<0.05). The Tafel curves of NiTi wires in two groups were moved to the left with the increasing of F^- concentration and the decreasing of pH value,which indicated that the corrosion resistance ability was decreased. Under scanning electron microscope, much pitting corrosion was observed on the surface of NiTi wires in control group than experiment group. Conclusion: The corrosion resistance ability of DLC coated NiTi wires is higher than that of uncoated NiTi wires,but the increasing of F^- concentration and the decreasing of pH value can decrease the corrosion resistance ability of NiTi wires.

Objective: To investigate the stabilities of copy number of common reference genes in cell-free DNA in plasma of the pregnant women and non-pregnant women,and to provide the reference for the quantitative study of cell-free DNA in plasma of the pregnant women. Methods: Eighteen healthy pregnant women with twelve weeks of gestation and eighteen healthy non-pregnant women were investigated.The plasma cell-free DNA was extracted from the peripheral blood.All the DNA samples were divided into pregnant+non-pregnant group,pregnant group,non-pregnant group,maternal+fetal group,maternal group,and fetal group.The HBB,TERT,GAPDH,ALB,ACTB,and TRG were chosen as the reference genes.The Ct values of six reference genes in various groups were obtained by Real-time fluoresence quantitative PCR (qPCR) method.Three statistical softwares (geNorm,NormFinder and BestKeeper) were applied for evaluating the stabilities of copy number of six reference genes in various groups. Results: 1PCR products of all six kinds of reference genes were specific amplification by qPCR method.The differences of Ct values of reference genes in various groups were not statistically significant (P>0.05).The Ct value of ACTB in each group was the lowest,and followed by HBB,which meaned the ACTB and HBB had the highest copy number in the plasma cell-free DNA.2According to the descending order of average expression stability value (M) calculated by geNorm software,the descending order of stability value calculated by NormFinder software,and the descending order of coefficient of correlation value (R) calculated by BestKeeper software,the copy number stabilities of six reference genes were sorted in descending order in various groups.Comprehensive analysis of the results from three softwares,in pregnant+non-pregnant group,pregnant group,non-pregnant group,maternal+fetal group,maternal group, and fetal group,the genes with the highest copy number stabilities were TERT,ACTB,ALB,HBB,HBB,and HBB,respectively.3The differences of Ct values of reference genes between maternal+fetal group, maternal group,maternal group and fetal group were statistically significant (F=114.84,P<0.05). Conclusion: When performing quantitative analysis of plasma cell-free DNA of subjects in pregnant+non-pregnant group,pregnant group,non-pregnant group,maternal+fetal group,maternal group, and fetal group through qPCR method,TERT,ACTB,ALB,HBB,HBB and HBB are recommended respectively as the calibration reference genes.

Objective: To detect the expressions of metadherin (MTDH) and matrix metalloproteinase-9 (MMP-9) in breast cancer and paired adjacent normal breast tissues of the patients with basal-like breast carcinoma (BLBC) and to analyze their correlation. Methods: The expressions of MTDH and MMP-9 in breast cancer and paired adjacent normal breast tissues of 60 BLBC patients were detected by immunohistochemistry method.Pearman analysis was used to detect the correlation between the positive expression rates of MTDH and MMP-9. Results: The positive expression rates of MTDH and MMP-9 in breast cancer tissue were higher than those in paired adjacent normal breast tissue(P<0.05).The expressions of MTDH and MMP-9 in breast cancer tissue were positively correlated with lymph node metastasis and TNM staging of the BLBC patients (P<0.05).The positive expression rates of MTDH and MMP-9 in the BLBC patients with positive lymph node metastasis were much higher than those in the BLBC patients without lymph node metastasis (χ²=6.89,P<0.05;χ²=15.22,P<0.01);the positive expression rates of MTDH and MMP-9 in the BLBC patients in Ⅲ+Ⅳ stage were much higher than those of the BLBC patients in Ⅰ+Ⅱ stage(χ²=6.431,P<0.05;χ²=8.014,P<0.05).There was a positive correlation between the expression of MTDH and the expression of MMP-9 in breast cancer tissue of the BLBC patients (r=0.531,P<0.01). Conclusion: The expression of MTDH is positive correlated with the expression of MMP-9 in breast cancer tissue of the BLBC patients;furthermore,MTDH and MMP-9 protein may play an important role in the carcinogenesis and development of BLBC.

Objective: To evaluate the role of the multiple tumor markers protein chip (C12 chip) in healthy screening of old people,and to quantitatively analyze the changes and clinical significance of the detection values and positive rates of tumor markers of the subjects with different ages and genders. Methods: A total of 9 654 cases were selected. According to the age,the subjects were divided into control group(aged 40 to 59),old people group(aged 60 to 79)and senile people group(aged 80 and above).The protein chip system for multiple tumor marker detection(Chip C-12) was used to measure the levels of 12 tumor markers including alpha fetoprotein(AFP),carcinoembryonic antigen(CEA),carbohydrate antigen 15-3(CA15-3),carbohydrate antigen 19-9(CA19-9),carbohydrate antigen 125(CA125),carbohydrate antigen 242(CA242),ferritin,human chorionic gonadotropin beta(β-HCG),human growth hormone(HGH),neuron specific enolase (NSE),prostate special antigen(PSA) and free PSA(f-PSA).The positive result was confirmed when the levels of one or more tumor markers were higher than the reference value.The detection values and the positive rates of the subjects in three groups were analyzed. Results: 1Among 9 654 cases,the serum levels of tumor biomarkers were found to be positive in 665 cases,and the positive rate was 6.89%;among them 32 cases were diagnosed as early or intermediate cancer through endoscope,pathological and imageological diagnosis,and the detection rate was 0.33%.2 The positive rates were different in different genders. In male,the top five order was f-PSA,PSA,CEA,CA19-9, and NSE;while in female,the top five order was CA19-9,CA125,CEA,NSE and CA242.3positive rates of CA19-9,CA242,CEA,NSE,PSA and f-PSA were increased with the increasing of age(P<0.01).4The values of AFP,CA125,CA19-9,CA242,CEA,HGH,NSE,and PSA were increased with the increasing of age; the above indexes of the subjects in senile people group were higher than those in old people group and control group(P<0.01),and the above indexes of the subjects in old group were higher than those in control group (P<0.01). Conclusion: The multiple tumor markers (CA19-9,CEA,NSE,PSA,and f-PSA) of C12 chip are useful in the healthy screening of the old people, especial in the senile people to discover tumor at an early stage.

Objective: To explore the effect of physical constitution on the serum cardiac troponin I(cTnI) level in the children with pneumonia complicated by heart failure,and to assess its clinical value. Methods: Atotal of 116 patients aged 1-3 years with pneumonia complicated by heart failure were enrolled in this study as observation group,and 98 healthy children aged 1-3 years were enrolled as control group.The height and weight of each subject were measured,and the infant Kaup index was calculated with these parameter.The subjects in two groups were divided into 3 subgroups according to the Kaup index: normal group (Kaup index 13.5-18.0 kg·cm^-2),excellent group(Kaup index 18.1-20.0 kg·cm^-2),and obese group(Kaup index>20.0 kg·cm^-2).The levels of serum cTnI and hsCRP of subjects were detected, respectively,and the relationship between the levels of serum cTnI,hsCRP and Kaup index was analyzed. Results: The levels of serum cTnI and hsCRP of the subjects in observation group were significantly higher than those in control group (P=0.000),and they were increased with the increasing of Kaup index,and they were positively correlated with Kaup index(r=0.555,P=0.000;r=0.705,P =0.000).The level of serum cTnI was positively correlated with the level of serum hsCRP(r=0.407,P=0.000). Conclusion: In the children with pneumonia complicated by heart failure,the serum cTnI level is affacted significantly by physical constitution,and the levels of serum cTnI and hsCRP are high if the Kaup index of subject is high,so relying only on the level of serum cTnI is not conducive to the diagnosis and prognosis for the obesity patients with pneumonia complicated by heart failure.

Objective: To compare the expression levels of Fos-like antigen 2 (FOSL2) mRNA of the patients with type 2 diabetes mellitus(T2DM) and the subjects with nomal glucose tolerance (NGT) in Uygurs in Xinjiang,and to analyze the correlation between the FOSL2 mRNA expression and the clinical and biochemical indicators of the subjects. Methods: A total of 50 Uygrus adult patients with T2DM(T2DMgroup) and 50 cases of NGT (NGT group) were collected respectively in Kashi area in Xinjiang.The general clinical indicators,such as gender,age,pulse,systolic blood pressure(SBP),diastolic blood pressure(DBP),WHR,BMI, and T2DM disease course identified by the diagnostic criteria of WHO were recorded,and the blood samples were collected at the same time.The metabolic markers such as fasting blood glucose(FPG) total cholesterol(TC),TG,LDL-C and HDL-C were measured by clinical chemistry analyzer;the level of fasting serum insulin (FINS) was measured by automatic chemiluminescence analyzer;the level of HbAlc was mesured by high pressure liquid chromatography(HPLC);the expression levels of FOSL2 mRNA in leukocytes of the subjects were examined by RT-PCR method. Results: Compared with NGT group,the BMI(t=-4.62,P=0.00),level of FPG(t=-9.56,P=0.00),HbA1c(t=-7.83,P =0.00),TC(t=-7.86,P =0.00),TG(t=-3.89,P=0.00),and LDL-C(t=-2.41,P =0.01) were increased;however,the level of HDL-C(t=-2.58,P=0.01)and FOSL2 mRNA expression level(t=-12.174,P =0.00)in leukocytes of the patients in T2DM group were decreased. Compared with NGT group,the FINS(t=-2.42,P=0.02),homeostasis model assessment of insulin resistance(HOMA-IR) (t=-6.46,P=0.00),nataral logarithm homeostasis model assessment of insulin β-cell function(In-HOMA-β) (t=5.58,P=0.00) of the patients in T2DM group were increased ,and the nataral logarithm insulin sensitivity index(In-ISI) (t=6.80,P=0.00) was decreased. WHR(r=-0.415,P=0.008),and the levels of FPG(r=-0.620,P=0.000),HbAlc(r=-0.681,P=0.000),HOMA-IR(r=-0.504,P=0.001),TC(r=-0.582,P=0.000),TG(r=-0.342,P=0.031),T2DM disease course (r=-0.733,P=0.000) and HDL-C(r=-0.323,P=0.042)were negatively correlated with the expression level of FOSL2 mRNA in leukocytes of the T2DM patients. The level of In-HOMA-β(r=0.638,P=0.000) showed a positive correlation with the expression level of FOSL2 mRNA. The results of regression analysis showed that T2DM disease course(β=-0.056,P=0.002),In-HOMA-β(β=0.342,P=0.001),and TC(β=0.219,P=0.003)were the influencing factors of the level of FOSL2 mRNA in the T2DM patients. Conclusion: The down-regulation of expression level of FOSL2 mRNA may be involved in the occurrence and development of insulin resistance and T2DM in Uygurs in Xinjiang.TC is an important factor of the down-regulation of expression level of FOSL2 mRNA in the Uggurs patients with T2DM.

Objective: To detect the expression levels of IL-22 mRNA in peripheral blood mononuclear cells (PBMCs) of the patients with chronic hepatitis B (CHB),acute/subacute-on-chronic liver failure (severe hepatitis),and hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC),and to investigate their clinical significances. Methods: A total of 20 healthy controls (control group),33 patients with moderate CHB(moderate CHB group),21 patients with severe CHB(severe CHB group),16 patients with severe hepatitis(severe hepatitis group),16 patients with cirrhosis (cirrhosis group) , and 40 patients with HCC(HCC group) were enrolled.The expression levels of IL-22 mRNA in PBMCs of the subjects in various groups were tested by RT-PCR method;Pearson correlation analysis was used to analyze the relationship between the IL-22 mRNA expression levels and the important clinical indexes. Results: The expression levels of IL-22 mRNA in PBMCs of the patients in moderate CHB,severe CHB and cirrhosis groups were all higher than that in control group(P<0.001);there were no significant differences of the expression levels of IL-22 mRNA of the subjects between severe hepatitis group and control group (P=0.83);the expression level of IL-22 mRNA in PBMCs of the patients in HCC group was lower than that in control group (P<0.001);the expression level of IL-22 mRNA in PBMCs was negatively correlated with the serum total bilirubin(TB) level in severe CHB and severe hepatitis groups(r=-0.383,P=0.043;r=-0.619,P=0.028);the expression level of IL-22 mRNA was positively correlated with the serum alpha-fetoprotein (AFP) level in HCC group (r=-0.664,P=0.000). Conclusion: The decreasing of IL-22 mRNA expression level in the patients with deteriorative hepatitis suggests the dysfunction of liver regeneration;the IL-22 mRNA expression level,as TB,can reflect the severity of liver injury. IL-22 may have promotion effect on the occurrence and development of HCC.

Objective: To detect the levels of serum thymic stromal lymphopoietin(TSLP) in the patients with rheumatoid arthritis(RA) or systemic lupus erythemtosus (SLE) with joint symptoms,and to clarify the possible mechanisms of TSLP in the process of RA and SLE. Methods: A total of 40 patients with RA were chosen as RA group,20 SLE patients with joint symptoms were chosen as SLE group,and 20 healthy people were selected as normal control group.The expression levels of serum TSLP of the subjects in various groups were examined by ELISA method and the clinical activity indexes of RA patients were collected,such as C-reaction protein(CRP),erythrocyte sedimentation rate(ESR),cyclic citrullinated peptide(CCP),tender joints count(TJC),swollen joints count(SJC),visual analogue scale(VAS) and disease activity score(DAS28-ESR,DAS28-CRP);the clinical symptoms of SLE patients were collected,such as arthritis,new rash,ulcer and other symptoms; the clinical activity indexes,such as routine blood,24 h urine protein etc.and systemic lupus erythematosus disease activity index(SLEDAI) were estimated.Pearson test was used to analyze the correlation between the serum TSLP level and the indexes of the patients in RA and SLE groups. Results: The level of serum TSLP in the patients with RA was higher than that in normal control group(t=2.73,P<0.05).The level of serum TSLP in the patients with RA was positive correlated with CRP,ESR and anti-CCP antibodies(r=0.504,P=0.001;r=0.550,P<0.0001;r=0.895,P<0.0001); the level of serum TSLP had no correlation with DAS28-ESR and DAS28-CRP(r=0.213,P=0.187;r=0.170,P=0.293).The level of serum TSLP in the patients with positive anti-CCP antibody was higher than that in the patients with negative anti-CCP antibody(F=16.99,P<0.05).The level of serum TSLP in the patients with SLE had no significant difference compared with normal control group(t=0.673,P>0.05),and it had no correlation with SLEDAI(r=0.079,P=0.749). Conclusion: The serum level of TSLP in the patients with RA is obviously increased and TSLP may participate in the pathogenesis of RA and correlate with disease activity.TSLP may be involved in the regulation of anti-CCP antibodies.The changes of serum TSLP level in the RA patients have a certain reference value in monitoring the therapeutic effects.

Objective: To detect the ratios of peripheral blood Th9 cells and the levels of serum IL-9 in the patients with small cell lung cancer (SCLC),and to investigate the effect of Th9 cells on the development of human SCLC. Methods: Twenty SCLC patients at limited stage(limited stage SCLC group),16 SCLC patients at extensive stage(extensive stage SCLC group), and 10 healthy controls (healthy control group) were selected.The ratios of Th9 cells to CD4⁺ cells in peripheral blood and the levels of serum IL-9 were detected by flow cytometry and ELISA, respectively. Results: Compared with healthy control group,the level of serum IL-9 of the patients in SCLC group was increased(t=4.001,P<0.01),and the levels of IL-9 of the patients in limited stage SCLC group was lower than that in extensive stage SCLC group(t=3.089,P<0.01).The ratio of Th9 cells of the patients in SCLC group was higher than that in healthy control group (t=3.731,P<0.01),and the ratio of Th9 cells of the patients in limited stage SCLC group was lower than that in extensive stage SCLC group (t=2.825,P<0.01). Conclusion: Th9 might directly promote the development of SCLC through secreting IL-9.The peripheral blood Th9 cells and IL-9 might be biomarkers to evaluate SCLC progression.

Objective: To establish the expended vertebra interbody fusion cage (EVIFC) and finite element analysis of L4-5 and to analyze the influence of EVIFC insertion in the biomechanical parameters of lumbar spine,and to provide the biomechanical evidence for its clinical application. Methods: According to the umbar CT scan data of healthy adult male people, the normal L4-5 lumbar spine finite element model was established.In this model,the L4-5 intervertebral disc slight degeneration was simulated,and the 3D model was implanted with EVIFC.Under flexion,lateral bending,and rotation loading conditions,the stability of lumbar spine model before and after EVIFC implantion,the pressure of the intervertebral disc,endplate pressure and small joint loading changes were analyzed. Results: After the insertion of EVIFC,the stress in end-plate,nucleus and small joint of the L4-5 level was decreased. In flexion,the mean pressure values in the end-plate and nucleus of the degeneration model were 6.162 0 and 0.201 6 MPa,and the small joint load was 32.8 N.After implantation of EVIFC ,they were 3.526,0.107 MPa,and 8.5 N,respectively.The consistent results occurred in another two loading conditions,As for the adjacent levels,compared with degeneration model,the stress in end-plate and nucleus were decreased at the L4-5 level.The mean stress in the annulus of L4-5 was increased slightly. Conclusion: EVIFC has effect on the stability of slightly degenerative lumber spine which can meet the needs of human biomechanics,and it is a suitable and new lumbar fusion device.

Objective: To investigate a safe and convenient way to perform an enblocresection of giant solitary fibrous tumor of pleura (SFTP). Methods: The clinical data of 5 patients with giant SFTP(the diameter>10 cm) were collected. Enhanced CT method was used to search the nutrient artery of tumor.Thoracoscope was used to select the location and diameter of the tumor and to assist the separation from the important organ and exposure the operation field,so as to resect the tumor completely.The operation method,diameter of the tumor,operation time,blood loss,drainage time,incisal margin and postoperative complications were recorded. Results: The tumor of 5 patients were all removed.The mean operation time was 112 min.The blood lose during the operation was 210 mL.The mean drainage time was 3.8 d.There were no perioperative death.There was no tumor recurrence found in the 6-36 months follow up. Conclusion: For the SFTP patients, an enhanced CT and artery embolization preoperatively should be perfomed to judge the blood supplyment of tumor and its relationship with surrounding organs.Thoracotomy assisted by thoracoscope may offer a safe and feasible method to accomplish an enblocresection.

Objective: To report a case of gout arthritis in the second metatarsophalangeal joint complicated with congenital brachydactyly,and to discuss the diagnosis and treatment conditions. Methods: One male patient aged 17 years old manifested as repeated pain and swelling in the second metatarsophalangeal joint for 6 months. The diagnosis result showed gout arthritis in the second metatarsophalangeal joint complicated with congenital brachydactyly. Gout stone excision and arthroplasty were performed to relieve the symptoms. Results: The patient recovered without complications.The pain disappeared postoperatively.The patient returned to the normal walking function. Conclusion: The gout and congenital brachydactyly can be presented in one patient.Differential diagnosis is essential for the case,and the surgical excision is curative.

Objective: To discuss the experiences and therapeutic effects of Viabahn endovascular treatment in transluminal vascular occlusive disease of long segment of iliac-femoral artery,and to evaluate its curative effect in occlusive disease of peripheral artery. Methods: The clinical data of total 78 patients (89 lower limb) with transluminal vascular occlusive disease of long segment of iliac-femoral artery were collected and the patients were divided into bare stent group (42 cases,48 lower limb) and Viabahn group (36 cases,41 lower limb).The general information,peri-operative and long-term patency rate,and rate of limb salvage of patients in two groups were collected.The follow-up time was 3-12 months and the average follow-up time was 11.08 months. Results: Compared with before operation,the ischemic symptoms of the patients in two groups after operation were improved and the ankle brachia index(ABI) was increased(P<0.05).3,6,and 9 months after post-operative follow-up,the primary and secondary patency rates of the patients in two groups showed no significant difference (P>0.05).12 months after post-operative follow-up,the primary and secondary patency rates of the patients in Viabahn group were higher than those in bare stent group (P<0.05).The 1-year limb salvage rates of patients in two groups were 100%.The patients in bare stent group had more risk to suffer stent thrombosis than those in Viabahn group (P<0.05). Conclusion: Using Viabahn for treatment of transluminal vascular occlusive disease of long segment of iliac-femoral artery (≥10 cm) is safe and effective,and the 12-month long-term patency rate and the risk to suffer stent thrombosis are superior to bare stent.

Objective: To observe the effect of botulinum toxin A injection treatment on the spasticity of triceps surae and walking ability of the patient with stroke,and to discuss the relationship between the muscle tension changes and motor function of lower limb and the feasibility of its application in the community rehabilitation. Methods: Thirty stroke patients with triceps surae spasticity were divided into treatment group(n=15) and control group(n=15) according to the patient's will.In treatment group,the botulinum toxin A were injected into the triceps surae of patients before routine physical therapy,while the patients in control group only received routine physical therapy,including muscular stretching,treatment of muscle strength,balance function and walking ability.The modified Ashworth scale (MAS),Fugl-Meyer Assessment (FMA) and 10-meter walk test (10MWT) time were used to detect the ability of lower limb.All the test was performed before,after,and 3 months after the treatment. Results: The MAS scores of the patients in treatment group after treatment and 3 months after treatment were lower than that before treatment (P<0.05);there was no significant difference of MAS scores of the patients in control group after treatment(P>0.05),and the MAS score of patients 3 months after treatment was lower than that before treatment(P<0.05).The FMA score and 10MWT time of the patients in two groups after treatment and 3 months after treatment were higher than those before treatment(P<0.05).Compared with control group,the MAS scores and 10MWT time of the patients in treatment group after treatment and 3 months after treatment were decreased and the FMA scores were increased(P<0.05). Conclusion: The botulinum toxin A treatment combined with routine physical therapy may improve the spasticity of triceps surae,gait,and walking ability in a better way,which can be applied in the community rehabilitation.

Objective: To investigate the image characteristics of ¹⁸F-fluorodeoxyglucose (¹⁸FDG) positron emission tomography and computer tomography(PET/CT) in the patients with dermatomyositis (DM) or polymyositis (PM)associated with malignancy,and to provide basis for the diagnosis of DM/PM. Methods: The clinical data of inpatients diagnosed with DM/PM during 2009 to 2015 in Peking Union Medical College Hospital were collected.The clinical manifestations and ¹⁸FDG PET/CT wholebody scan were analyzed in the 17 patients with DM/PM complicated with tumor and 39 patients with DM/PM without tumor. Results: Among 56 patients with DM/PM,17 patients were complicated with maglignant tumor; there were 5 patients with lymphoma, 7 patients with lung cancer, 1 patient with thyroid carcinoma, 1 patient with breast cancer,1 patient with cervical carcinoma, 1 patient with neuroendocrine tumor of liver,and 1 patient with thymoma.The maximal standard uptake value(SUVmax) of malignant lesions was 1.47-22.93(7.67±6.60).There was diffuse increase of ¹⁸FDG uptake in the muscles in 22 patients,and the SUVmax was 0.71-2.93(1.43±0.68); there were 9 patients(40.91%)associated with malignancies.There was local increase of ¹⁸FDG uptake in the muscles in 26 patients,and the SUVmax was 1.06-8.74(2.62±1.65). There were 6 patients(23.08%)associated with malignancies.There was normal level of ¹⁸FDG uptake in the muscles in 8 patients; There were 3 patients(37.50%)associated with malignancies. There was 15 patients with interstitial pneumonia,and 2 patients(13.33%)associated with malignancies. Conclusion: The ¹⁸FDG PET/CT image of tumor-associated DM/PM has the certain characteristics.The patients with diffuse increase of ¹⁸FDG uptake in the muscles have the highest incidence rate of tumor.¹⁸FDG-PET/CT may become an effective method of assisted diagnosis for the patients with suspected DM/PM.

Objective: To locate the surface projection of transverse sinus on the skull using the connecting line between inion and low point of mastoid,and to provide the morphological foundation for infratentorial-supracerebellar approaches and other related approaches and to protect the transverse sinus. Methods: Eighty adult cases were scanned with thin-section CT.The skull 3D model was rebuilt by CT reconstruction technology for the marking of structures and measurements.The connecting line between the inion(A) and the low point of mastoid (B) was chosen as the marker line,and the morphological parameters of distances and projection from the groove of the transverse sinus to the connecting line were measured. Results: The width of marker point J in middle of transverse sinus on the right hemisphere was bigger than that on the left hemiphere (t=5.232,P<0.05);there were no statistical differences in the measurement values of h1,h2,h3,h4,h5,s1,s2,BE,BD,BJ, and BI from the marker points of groove of transverse sinus to the connecting line AB between two cerebral hemispheres(P>0.05);there were no statistical differences in the measurement values of h1,h2,h3,h4,h5,s1,s2,BE,BD,BJ,and BI from the marker points of groove of transverse sinus to the connecting line AB between genders (P>0.05). Conclusion: The surface projection of transverse sinus is obtained accurately with CT reconstruction image.The method is simple and accessible which can guide to protect transverse sinus during the operation.

Objective: To summarize the echocardigraphic characteristics of dynamic intraventricular obstruction (DIVO) of the left ventricle by traditional echocardiography at rest and to design a new index(R index,V_IVR/V_E) according to its characteristics,and to explore the value of R index in quantitative evaluation on DIVO. Methods: One hundred and twelve patients with DIVO were chosen as obstruction group. All patients had chest pain or dyspnea.A total of 50 healthy volunteers were used as healthy control group.The left ventricular end-systolic diameter (LVESD),left ventricular end-diastolic diameter(LVEDD),upper interventricular septum thickness (U-IVS),middle interventricular septum thickness (M-IVS) and left ventricular ejection fraction(LVEF) were obtained by two-dimensional echocardiogram.The maximal systolic velocities and gradients at the left ventricular outflow tract (LVOT),mid-ventricle and the apex of left ventricle were obtained at rest in all patients using color Doppler ultrasound.The myocardial movement and the flow of left ventricle were observed at different aspects.Then the velocity of E wave,the narrowest inner diameter,the systolic velocity,the velocity of isovolumic relaxation (VIVR) and R index of the narrowest part were obtained.The receiver operating characteristic (ROC) curve was builded and the area under curve(AUC) was calculated.An index about confirmed the diagnosis of R index point was obtained.Moreover,the specificity and sensitivity of R index for the diagnosis of DIVO of the left ventricle at rest was explored. Results: The heart rate of patients with DIVO in obstruction group was faster than that in healthy control group and the blood pressure was higher than that in healthy control group (P<0.01).The 2D echocardigraphic characteristics of the DIVO patients showed that the myocardial movement of left ventricle was enhanced.The left ventricle diameter of the DIVO patients in obstruction group was smaller than that in healthy control group,and the shape of the left ventricle was irregular.As for Doppler,the normal flow velocity gradient in the left ventricular of the patients with DIVO was changed.A Doppler spectrum with peak value moving forward was found in obstruction group,and a IVR wave was seen,and the velocity of IVR wave was faster than healthy control group(P<0.01) and the R index was enlarged obviously(P<0.01). Conclusion: The R index of DIVO patients enlarges significantly,and R index ≥1 is the specific diagnosis indicator of the DIVO patients at rest.

Objective: To discuss the changes of blood flow and blood vessels of the patients with hyperthyroidism(Graves disease),and to clarify the value of three-dimensional color Doppler ultrasound in quantitative diagnosis of blood flow in the thyroid gland of the patients with Graves disease. Methods: A total of 55 cases of Graves disease (case group)and 38 cases of healthy volunteers (control group)were selected and all the subjects were examined with three-dimensional ultrasound. QLAB software was used to rebuild the storage images with three-dimensional for calculating vascular index (VI),flow index (FI),and vascular flow index (VFI).Pearson correlation analysis was used to analyze the relationships between VI,FI,VFI in thyroid and the thyroid function indexes three free iodine thyroid original amino acid(FT₃),serum free thyroxine(FT₄),thyroid stimulating hormone(TSH)and the highest value of 24-hour iodine absorption rate of the patients in case group. Results: The differences of VI,FI,and VFI on the corresponding part of both sides of thyroid lobe of the patients in case group had no statistical significance (P>0.05);the differences of VI,FI,and VFI on the corresponding part of same side of thyroid lobe of the patients in case group had no statistical significance (P>0.05);the VI,FI,and VFI on the corresponding part of same side of thyroid lobe of the patients in case group were higher than those in control group(P<0.01);the VI(r= 0.65,P<0.01),FI(r=0.34,P<0.05),and VFI (r=0.65,P<0.01)in the left lobe of thyroid of the patients in case group were positively related with FT₃;the VI,FI,and VFI in the left lobe of thyroid of the patients in case group were positively related with FT₄(r=0.45,r=0.47,r=0.46,P<0.05);the VI and VFI in the left lobe of thyroid of the patients in case group were negatively related with TSH(r=-0.51,r=-0.54,P<0.05);the FI in the left lobe of thyroid of the patients in case group had no relationship with TSH(r=0.31,P> 0.05); the VI,FI,and VFI in the left lobe of thyroid of the patients in case group were positively related with the highest value of 24-hour iodine absorption rate (r=0.67,r=0.68,r=0.69,P<0.05). Conclusion: The three-dimensional color Doppler ultrasound can intuitively,stereoscopically and quantitatively evaluate the blood flow changes in thyroid of the Graves disease patients.The blood flow in thyroid of the Graves disease patients appears diffusivity increasing.

Objective: To investigate the prevalence of cyberbullying and related influencing factors in some college students in China,and to provide scientific basis for improving healthy network behaviors and promote college students' physical and mental health. Methods: A cross-sectional study from 15 January to 5 March in 2015 was performed in order to collect the information of social networking site usage of some college students in 27 provinces,autonomous regions,and municipalities.781 questionnaires were effective and used as the samples in the research.The questionnaire contained demographic information,social networking site situation, cyberbullying and psychological scale.According to the cyberbullying situation,all of the samples were divided in to cyberbully-only group,cybervictim-only group,and cyberbully-victim group.The incidence of cyberbullying of the college students in various groups was analyzed with χ² test. Wilcoxon Mann-Whitney test was used to analyze the incidence of cyberbullying of the students with different ages,frequency of logging in social networks,time spending on social networks,proportion of acquaintances,and number of friends. Results: In the total 781 samples,306 students (39.18%) participated in the cyberbullying. 53 students (17.32%) had cyberbullying only,and 111 students(36.27%) were cybervictims only,and 142 students(46.41%) were cyberbully-victims.416 students (53.27%) had identity crisis,and 419 students(53.65%) were not satisfied with their lives.The male,coming from west region,more than 2 times per day of using social networking sites,identity crisis,and satisfaction with life were the independent influencing factors of cyberbullying(OR=2.75,95%CI: 1.97-3.83;OR=3.09,95%CI: 1.883-5.07;OR=3.22,95%CI: 1.14-9.08;OR=1.55,95%CI: 1.11-2.16;OR=1.47,95%CI: 1.05-2.06). Conclusion: The incidence of cyberbullying in some college students in China is high. In order to reduce the cyberbullying,strengthening the management of college student's internet usage and improving their psychological health level are of great significance.

Objective: To optimize the liposome-mediated transfection conditions based on transfecting the pEGFP-N1 into thyroid cells of Fischer rats,and to investigate its effectiveness on other target gene transfection. Methods: Based on the different ratios and quantities of plasmids and liposomes,the thyroid follicular epithelial cells of Fischer rats were transfected with pEGFP-N1.The expression of enhanced green fluorescence protein(EGFP) and transfection efficiency were observed under inverted fluorescence microscope and flow cytometry, respectively.Then the cell viability was detected by Trypan Blue stainning.Meanwhile the thyroid follicular epithelial cells of Fischer rats were transfected with pEGFP-Aquaporin2,pEGFP-Aquaporin4 and pEGFP-Aquaporin5 under the above conditions,and the optimized conditions of transfecion were obtained. Results: When the quantity of plasmid was certain,the transfection efficiency and cell viability were increased with the increasing of liposomes,but the transfection efficiency and cell viability were decreased when the ratio of plasmid to liposome was over 1:4.When the ratio of plasmid to liposome was certain and the quantities were increased gradully,the transfection effciency was increased,and the cell viability was decreased.With the ratio of plasmids to liposomes was 1:4 (3.2 μg:12.8 μL),the transfecion efficencies of pEGFP-N1,pEGFP-Aquaporin2,pEGFP-Aquaporin2, and pEGFP-Aquaporin2 reached the highest values,respectively. Conclusion: The optimized conditions of transfection pEGFP-N1 is the same as the other plasmids, which can be used as optimized conditions for efficient transfection of the other target gene.

Objective: To prepare the sustained release microspheres of total flavonoids of chrysanthemum indicum(TFC)-polyactic-co-glycolic acid(PLGA) and to optimize the conditions. Methods: The TFC-PLGA microspheres were prepared by using the double emulsion solvent evaporation method.The effects of different conditions,such as the stirring rate,the polyvinyl alcohol(PVA) concentration and the ratio of TFC to PLGA on the entrapment efficiency(EE) of microspheres were observed.The general morphology of the microspheres was observed by microscope;the surface morphology and particle size of the microspheres were observed by SEM;the EE and in vitro release results of the microspheres were measured by UV spectrophotometry. Results: When the stirring rate was 3 000 r·min^-1,the PVA concentration was 3.0%,and the ratio of of TFC to PLGA was 1:15 as the optimal conditions,the acquired TFC-PLGA microspheres showed well-defined properties,with uniform sphere in appearance,general particles without adhesion,and the average EE was (45.03±1.25)% and the mean diameter was (102.20±1.97)μm.The results of release experiment in vitro showed that the percentage of accumulative release were 22.07% in 24 h and 92.32% on 20 d and the micropheres had significant sustained release effect. Conclusion: The TFC-PLGA microspheres with suitable size, uniform dispersion and high EE can be acquired under the optimized conditions.