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Objective To obtain recombinant adeno-associated virus capable of over-expressing OPTN and TAU-P301-L genes, to detect its infection of 293 T cellsin vitro, and to provide new ideas for the study of Alzheimer's disease(AD). Methods The plasmid containing the gene of interest was extracted from Escherichia coli containing the gene of interest, and the target gene was obtained by PCR amplification. The target gene OPTN was constructed intopAAV-IRES-ZsGreen1vector, and the target gene Tau-P301 L-V5 was constructed intopAAV-CMV-mkate-IRESvector. Plasmid positive clones were identified and sequenced. The recombinant plasmidspAAV-IRES-ZsGreen1-AAV9-OPTNandpAAV-CMV-mkate-IRES-AAV9-Tau-P301L-V5 were co-transfected into 293 T cells with the packaging plasmidpAAV-RC9 and the helper plasmidpHelperby LipofiterTM2000, respectively, to obtainAAV9 virus. Using the same method, the corresponding green and redAAV9 control virus were constructed without using the target gene.The titer of recombinant adeno-associated virus was determined by qPCR method. The virulence and safety of 293 T cells were detected by viral vector, and the protein expression was identified by Western blot and RT-PCR. MTT assay was used to detect the effect of OPTN protein and TAU-P301-L protein on the activity of 293 T cells. Results The sequencing results showed that theAAV-IRES-ZsGreen1-AAV9-OPTNandpAAV-CMV-mkate-IRES-AAV9-Tau-P301L-V5 adeno-associated virus vectors were successfully constructed. The virus titer determined by qPCR was 1.0×1012vg/ml. The transfection efficiency of virus vector-infected 293 T cells was 33.6% and 20.5%. Western blot and RT-PCR showed protein overexpression. MTT results showed that the OPTN group rescued the apoptosis of the TAU-P301-L group. Conclusion Adeno-associated virus over-expressing OPTN and TAU-P301-L genes was successfully constructed. OPTN protein can reduce the cytotoxicity of TAU-P301-L protein.

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Objective To investigate the mechanism of gliclazide in improving hepatic insulin resistance of type 2 diabetes mellitus(T2 DM). Methods We established T2 DM animal model by high fat diet combined with intraperitoneal injection of streptozotocin. Fasting blood glucose, serum insulin, glucose tolerance, insulin sensitivity and liver pathology in T2 DM mice were detected. Glucose consumption and glycogen content were measured. The expression of insulin signaling pathway proteins(insulin receptor, insulin receptor substrate1, phosphatidylinositol 3-kinase, glycogen synthesis kinase 3β and glucose transporter 4) in liver tissues and HepG2 cells were detected by WB. Results Compared with T2 DM group, the mice in gliclazide groups showed decreased blood glucose, increased serum insulin, significantly improved glucose tolerance and insulin sensitivity, and reduced hepatocyte necrosis and vacuolation. Gliclazide significantly increased glucose consumption and glycogen content in insulin resistant HepG2 cells. Gliclazide increased insulin receptor, phosphatidylinositol 3-kinase and glucose transporter 4 expression and insulin receptor substrate1 tyrosine phosphorylation. Gliclazide inhibited glycogen synthesis kinase 3β tyrosine phosphorylation. Conclusion Gliclazide can improve glucose metabolism disorder, insulin sensitivity and liver injury in T2 DM mice. Gliclazide can increase glucose consumption and glycogen content in insulin resistant HepG2 cells. The mechanism may be related to the up-regulated insulin signaling pathway.

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Objective To investigate the inhibitory effect of MTAN on peri-implant inflammatory bacteria and biofilm. Methods Different concentrations of Nisin MTAN, MTAN+EDTA were used to treatP.gingivalis,F.nucleatumandS.sanguinis. Chlorhexidine and sterile water were used as negative and positive controls, respectively. The OD value was used to measure MIC and MBC. Biofilm ofF.nucleatumwas established by cementum sheet. Biofilm morphology was observed by scanning electron microscopy under different concentrations of the reagents and effect. Results MTAN forS.sanguinis,F.nucleatum,P.gingivalis, of MIC were 31.3,62.5 and 125 μg/ml while MBC were 125,250,500 μg/ml. MTAN+EDTA MIC of three kinds of bacteria were 31.3,31.3,62.5 μg/ml while MBC were 125,62.5,250 μg/ml. OD values displayed above the MIC of each group were significantly different(P<0.05) with the negative control group. Conclusion Both MTAN and MTAN+EDTA had strong inhibitory effects on the pathogenic bacteria of peri-implant inflammation, among which MTAN+EDTA had stronger inhibitory effects onP.gingivalisandF.nucleatum. The inhibition of MTAN and MTAN+EDTA on the biofilm formation ofF.nucleatumincreased with the increase of Nisin concentration.

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Objective To investigate regulatory T cell(Treg), including CD8+Treg and CD4+Treg, and their relationship with NK cells in patients with hepatocellular carcinoma(HCC). Methods A total of 55 patients with HCC and 22 healthy controls were collected. Peripheral blood and liver tissues were collected from HCC patients. Peripheral blood samples were obtained from healthy controls. CD8+CD25+Treg, CD4+CD25+Foxp3+Treg and NK cells were detected by flow cytometry. Results The levels of CD8+regulatory T cells in the peripheral blood of healthy people and patients with HCC were measured, and the CD8+CD25+regulatory T cells in the peripheral blood of the patients(6.22±0.49)% were higher than those in the healthy people(4.56±0.44)%(P<0.05); The proportion of CTLA-4+CD8+regulatory T cells in cancer tissues(41.00±4.00)% was higher than that in adjacent cancers(6.23±2.75)%(P<0.05). Further comparison of CD4+regulatory T cells in cancerous tissues and adjacent cancerous tissues revealed that the proportion of CD4+CD25+FoxP3+regulatory T cells in cancerous tissues(23.28±6.78)% was higher than that in adjacent cancerous tissues(10.18±4.04)%(P<0.05). The proportion of CTLA-4+CD4+Treg cells in cancerous tissue(61.53±11.37)% was also significantly higher than that in adjacent cancerous tissue(16.33±2.38)%(P<0.05). Analysis of NK cell expression revealed that the expression ratio of NK cells in the HCC group(13.57±1.39)% was significantly lower than that in the healthy group(21.44±1.23)%(P<0.05); the proportion of NK cells in cancer tissues(7.29±10.82)% was also lower than that in adjacent cancerous tissue(25.65±3.67)%(P<0.05). The expression of NK cells in peripheral blood of HCC patients was negatively correlated with the expression of CD8+regulatory T cells(r=-0.66,P<0.05); the proportion of CD4+regulatory T cells in cancer tissue was also negatively correlated with the expression ratio of NK cells(r=-0.76,P<0.05). Conclusion The proportions of CD8+Treg and CD4+Treg with increased CTLA-4 expression in HCC patients were detected. The percentage of NK cells in PBMC and cancer tissues from HCC patients was significantly lower than that of adjacent cancerous tissues and healthy controls. The frequencies of CD8+Treg and CD4+Treg were negatively correlated with NK cells. Our results indicate that CD8+and CD4+Tregs mediate suppressive inhibition of NK cell function in HCC patients, suggesting targeting CTLA-4+Treg mediated suppression contribute to restored NK cell function.

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Objective To investigate the effect of legumain knockout and icaritin on bone metastasis in breast cancer. Methods Wild-type and legumain knock-out mice were operated for heart injection, and the therapy group was treated with icaritin. Micro-CT was used to detect bone erosion caused by bone metastasis in breast cancer. Western blot experiments were used to detect relevant indicators. Results Mice with legumain knock out alone could slightly alleviate bone metastasis in breast cancer. Combined with icaritin,they could effectively alleviate bone metastasis in breast cancer via affecting signal transducer and activator of transcription-3(STAT3), glycogen synthase kinase-3β(GSK3β), β-catenin pathways. Conclusion Legumain knockout cooperated with icaritin can effectively relieve breast cancer bone metastasis.

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Objective To investigate the expression of long noncoding RNA(LncRNAs) HOTAIR in acute myeloid leukemia(AML) and the function and molecular mechanism of resistance to daunorubicin(DNR) in AML cells. Methods The qRT-PCR was used to detect leukemia cell lines, primary acute myeloid leukemia cells, the normal expression level of control cells HOTAIR. U937 cells and THP1 cells were selected as research objects.The sh-NC plasmid and sh-HOTAIR plasmid of hairpin RNA(shRNA) were constructed and transfected into these two cells, respectively. The expression of HOTAIR in the cells was detected by qRT-PCR. P-glycoprotein(P-gp) expression in transfected cells was detected by Western blot,DNR(0.1 μmol/L) was applied to each cell, CCK8 assay was used to detect cell proliferation, and flow cytometry was used to detect cell apoptosis. Results The expression of HOTAIR in the normal control group was significantly lower than that in the leukemia cell line and the acute myeloid leukemia primary cells; The expression of HOTAIR in U937 cells and THP1 cells was successfully down-regulated, and the level of P-glycoprotein was also decreased. The expression of HOTAIR in U937 cells and THP1 cells was successfully down-regulated, combined with DNR was more conducive to inhibit cell proliferation and promote apoptosis. Conclusion The abnormal expression of HOTAIR in AML is involved in the formation of resistance to DNR.

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Objective To investigate the regulation effect of matrix hardness on hemodynamics of macrophages. Methods A matrix gel was prepared using polyacrylamide. The mouse macrophage cell line Raw264.7 was inoculated on 2 kPa and 20 kPa polyacrylamide matrix gels with different hardness, and the elastic modulus of the matrix gels with different hardness was measured using a nanoindenter. Fluorescence microscopy was used to detect the number of macrophages adhered to the matrix with different hardness and the number of focal spots. Confocal microscopy was used to detect the extension area of macrophages grown on different matrix gels. Results It was found that the hardness of the two matrices was(2.00±1.78)kPa and(20.00±4.0)kPa, respectively, which was consistent with the target hardness. On 2 kPa matrigel, the adhesion of macrophages was(23±15), while cell adhesion on 20 kPa matrigel increased significantly(32±5,P<0.05). Compared with 2 kPa matrigel(32±5), the adhesion spots of macrophages on 20 kPa matrigel(42±8) also increased(P<0.05), and the expansion area of macrophages on 20 kPa matrigel significantly increased(P<0.05). Conclusion Higher matrix hardness can promote the adhesion of macrophages and change the biomechanical characteristics of the cells.

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Objective To explore the role of glutamate transporter 2(EAAT2) in electroacupuncture preconditioning to reduce cerebral ischemia in mice. Methods 32 male C57 BL/6 mice were randomly divided into 4 groups(n=8): whole brain ischemia-reperfusion(BCCAO) group, electroacupuncture pretreatment(EA)+ BCCAO group, EAAT2 inhibitor dihydrokainate acid(DHK)+EA+BCCAO group, EAAT2 agonist ceftriaxone sodium(CXT)+ BCCAO group. In the BCCAO group, only the bilateral common carotid arteries were ligated. The DHK+EA+BCCAO group and the EA+BCCAO group were pre-treated with electroacupuncture once a day and 30 mins a time for 5 consecutive days. DHK 200 μg/kg was injected into the lateral ventricle before ischemia 1 h. In the CXT+BCCAO group, ceftriaxone sodium 600 mg/kg was injected intraperitoneally once a day for 5 consecutive days. After 24 h of preconditioning, global cerebral ischemia was performed for 20 mins. Neurobehavioral scores and HE staining were used to calculate apoptotic neurons in hippocampal CA1 area. Western blot was used to detect EAAT2 protein expression, and immunofluorescence double-label staining was used to detect EAAT2 and astrocyte activation markers(GFAP). Results Compared with the BCCAO group, the EA+BCCAO group and the CXT+BCCAO group had higher behavioral scores and decreased the number of apoptotic neurons(P<0.05). Compared with the EA+BCCAO group, the scores of neurobehavioral characteristics in the DHK+EA+BCCAO group decreased, and the number of apoptotic neurons increased(P<0.05). Western blot results: compared with the BCCAO group, the expression of EAAT2 protein in the EA+BCCAO group and the CXT+BCCAO group increased(P<0.05); compared with the EA+BCCAO group, the expression of EAAT2 protein in the DHK+EA+BCCAO group decreased(P<0.05). Immunofluorescence double-label staining: compared with the BCCAO group, the expression of EAAT2 and GFAP protein in the EA+BCCAO group and the CXT+BCCAO group increased(P<0.05), the expression of EAAT2 and GFAP protein in the DHK+EA+BCCAO group and the EA+BCCAO group decreased(P<0.05). Conclusion Electroacupuncture preconditioning can produce cerebral ischemic tolerance by regulating EAAT2 on astrocyte membrane.

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Objective To establish an in vitro human aortic endothelial cell(HAEC) calcification model and explore the effect of exogenous growth differentiation factor 11(GDF11) on calcification. Methods Low-concentration(10 mmol/L)/high-concentration(30 mmol/L) β-glycerol phosphate+100 nmol/L dexamethasone+50 μg/ml L-ascorbic acid were used to stimulate HAEC, and an effective calcification induction protocol was selected. Different concentrations(0, 30, 50, 100 ng/ml) of GDF11 were used at different concentrations(0, 30, 50, 100 ng/ml) to stimulate HAEC at different times(0, 8, 24, 48 h), and the effective concentration and time of GDF11 were selected; HAEC was divided into blank group, GDF11 group, calcification group, GDF11 + calcification group. Western blot was used to detect the expression of bone morphogenetic protein(BMP) 2, BMP4, and GDF11; cell immunofluorescence was used to detect the expression of GDF11; RT-PCR was used to detect the gene expression of Runx2, Osterix, and GDF11; Alizarin Red S staining was used to detect calcification deposition. Results (1) The expression levels of GDF11, BMP4, and BMP2 proteins in the high-concentration group were higher than those in the blank group(P<0.05); the Runx2, Osterix, and GDF11 gene expression levels in the high-concentration group were higher than those in the blank group(P<0.05); alizarin red staining showed that calcium deposition increased in the high-concentration group compared with the blank group;(2) Expression of BMP2, BMP4 protein decreased after pretreatment with 100 ng/ml GDF11 for 24 h(P<0.05));(3) Compared with the calcification group, the expression of BMP2 and BMP4 protein in the GDF11+calcification group decreased(P<0.05), and there was no significant difference between the GDF11 group and the blank group; compared with the calcification group, the calcification deposition of GDF11+calcification group was reduced, and there was no significant difference between the GDF11 group and the blank group; the gene levels of Runx2 and Osterix in the GDF11+calcification group were lower than those in the calcification group(P<0.05), but there was no significant difference between the GDF11 group and the blank group. Conclusion GDF11 can inhibit calcification of HAEC and may be associated with the inhibition of osteogenic differentiation in HAEC.

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Objective To analyze the effect of procaine(PCA) on the methylation status and expression level of Septin9, and to explore the molecular mechanism of PCA inhibiting the proliferation of colon cancer DLD-1 and HCT116 cells. Methods Different concentrations of PCA treated colon cancer cells at different times, and MTS method was used to detect cell proliferation activity. Flow cytometry and Transwell experiments were used to detect the effects of PCA on cell cycle, apoptosis and migration ability; BGS method and qRT-PCR were used to detect the methylation status and expression level of Septin9 before and after PCA treatment. Results PCA inhibited the proliferation of DLD-1 and HCT116 cells in a concentration-and time-dependent manner(P<0.05). Compared with the control group, PCA treatment blocked cell growth in G2/M phase, significantly induced apoptosis and inhibited cell migration(P<0.05). After PCA treatment, the degree of Septin9 methylation decreased, the expression of Septin9 mRNA increased, and the differences were statistically significant(P<0.05). Conclusion PCA can inhibit the proliferation and migration of colon cancer cells by reversing Septin9 methylation.

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Objective To investigate the effects of different concentrations of glucose on the energy metabolism of red blood cells(RBC) in vitro and its molecular mechanism. Method Fresh anticoagulant blood was collected from 6 healthy adults. The white blood cells and plasma were removed by centrifugation. After washing RBC three times with PBS respectively, the 6 people's RBC were gently mixed and divided into 4 groups. D-glucose was added to make a final concentration of 0, 6, 20, 30 mmol/L, respectively. After incubation at 37 ℃ for 0, 24 and 48 hours, some of the RBC suspension was taken out to detect eryptosis, ATP concentration, hexokinase(HK) activity, pyruvate kinase(PK) activity, reactive oxygen species(ROS) content and hemolysis rate by using flow cytometry and multi-function microplate reader. The experimental results were analyzed by one-way ANOVA. Results Compared with the 0 mmol/L group, after incubating RBC with glucose at 6, 20, 30 mmol/L for 24 h and 48 h,the HK activity,PK activity,and ATP content in the RBC all increased,and upward trend with increasing glucose concentration,and the difference was statistically significant( P<0. 05); while ROS content and decay rate decreased,and decreased with increasing glucose concentration,the difference was statistically significant( P<0. 05). After culturing erythrocytes with different concentrations of glucose( concentrations of 0,6,20,30 mmol/L) for 48 h,the ROS content and erythrocyte decay rate in RBC of different concentrations increased with the incubation time,compared with 0 h,the difference was statistically significant( P<0. 05); HK activity and PK activity gradually decreased,compared with 0 h,the difference was statistically significant( P<0. 05). The ATP content decreased in a fluctuating manner,and compared with 0 h,the difference was statistically significant( P<0. 05). Conclusion Short-term high-glucose culture of RBC in vitro not only strengthens the energy metabolism in RBC,but also reduces the oxidative stress and eryptosis. However,no glucose supply leads to gradually exhausted ATP and increased oxidative stress and eryptosis.

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Objective To investigate the mechanism of tanshinone ⅡA(TSA) regulating PTEN on the autophagy pathway PI3 K/AKT/mTOR after cerebral ischemia in rats. Methods Rat model of cerebral ischemia(MCAO) was established and treated with TSA. The experiment was divided into three groups:normal control group, MCAO+ saline(NS) group and MCAO+TSA group. Immunohistochemistry and Western blot were used to detect the protein expression. Results Compared with the control group, the expression of PTEN protein in the MCAO+TSA group decreased, the levels of PI3 K, AKT and mTOR increased, and the difference was statistically significant(P<0.05). Compared with the two groups in the surgery group, the PTEN protein in the TSA group decreased significantly compared with the MCAO group, while the expression levels of PI3 K, AKT and mTOR in the TSA group increased slightly compared with the MCAO group, and the difference was statistically significant(P<0.05). Conclusion By enhancing the expression of PTEN in rats with cerebral infarction, TSA can reduce the expression level of PI3 K/AKT/mTOR in autophagy pathway, reduce the inflammation of cerebral infarction cells, so as to protect the brain tissue of rats.

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Objective To investigate the effects of ropivacaine on brain cell apoptosis and learning ability in neonatal mice. Methods 60 newborn mice were randomly divided into three groups, control group:low dose group and high dose group(20 mices/group). Mice in low dose group and high dose group respectively by micro syringe was given intraperitoneally 0.5% ropivacaine 20 μl and 60 μl. Mice in the control group was injected with 60 μl saline. 6 mice in three groups after administration of 12 h were killed. Serum inflammatory factor serum(TNF-α, IL-1β) were analyzed by ELISA. The mRNA expression level of pro-inflammatory cytokines and apoptosis related protein was analyzed RT-PCR and Western blot. The apoptosis of cerebral tissue staining was analyzed by TUNEL. Another 60 old newborn rats were randomly divided into control group, low-dose group and high-dose group(20 mices/group). After treatment for 29 days, the ability of learning and memory of mice was analyzed by Morris level maze test. Results Compared with the control group, TNF-α and IL-1β mRNA and protein levels in the brain tissue and serum of the low dose group and high dose group significantly increased(P<0.05). Compared with the control group, the levels of Bax and Caspase-3 in the low-dose group and high-dose group significantly increased, and the level of Bcl-2 significantly decreased(P<0.05). Compared with the control group, the apoptosis of brain tissue in the low dose group and the high dose group increased significantly(P<0.05). Compared with the low dose group, the level of apoptosis in the high dose group increased more significantly(P<0.05). Compared with the control group, in 29-32 d, low dose group and high dose group mice escape latency was prolonged at 31 d and 32 d, the high dose group escape latency was higher than the low dose group(P<0.05). Conclusion Ropivacaine can induce inflammation and apoptosis in the brain tissue of neonatal mice, thereby affecting the learning and memory ability of mice.

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Objective To observe the effects of125I seeds brachytherapy on apoptosis of HCCC-9810 cholangiocarcinoma cells and Livin expression.Methods The study included 5 groups:blank control group, vacant shell group, and125I seeds group(0.5 mCiGy, 0.7 mCiGy and 0.9 mCiGy group). HCCC-9810 cholangiocarcinoma cells were cultured in vitro. RT-PCR was used to detect the expression of Livin mRNA in HCCC-9810 cholangiocarcinoma cells 48 h after treatment with125I seeds. The expression of Livin protein in the cells was indicated using Immunohistochemistry and Western Blot. Flow cytometry was performed to assess apoptosis by using annexin V-fluorescein isothiocyanate(Annexin V-FITC)/PI double staining. Results Compared with blank control group and vacant shell group, cell apoptosis rates of the three experimental groups were increased(P<0.05), also, the expression of Livin was significantly enhanced(P<0.05). Among the three experimental groups, the lowest expression of Livin along with the highest cell apoptosis rate were detected in 0.5 mCiGy125I seeds group; but the expression of Livin in 0.7 mCiGy125I seeds group was higher than that of another two groups, accompanying with the lowest cell apoptosis rate. However, there was no significant difference in Livin expression and apoptosis rate between blank control group and vacant shell group. Conclusion There is significant difference in ability to induce cell apoptosis among different doses of125I, which may be related to the ability to elevate Livin expression in HCCC-9810 cholangiocarcinoma cells. Radio-resistance may be related to the level of Livin(an apoptosis suppressor gene) gene expression in cholangiocarcinoma cells.

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Objective To investigate ATG5 regulates VSMC proliferation by mediating endothelial exosomes. Methods HUVECs were separated by type II collagenase digestion at 37℃ and then cultivated in DMEM/F-12 culture containing 10% FBS. Tissue explants adherent method was applied to obtain VSMCs. VSMCs was cultivated in DMEM/F-12 culture containing 20% FBS. The uptake of endothelial exosomes by VSMCs was tracked by high-content cell imaging system. SiRNA was used to knock down the expression of ATG5. Results HUVECs with high purity and good viability were isolated successfully by 0. 1% type Ⅱ collagenase digestion. The cell displayed typical cobblestone-like morphology under the inverted phase-contrast microscope. Immunofluorescence technique validated that the isolated cells with CD31/Pan-Cadherin/Factor Ⅷ-positive phenotype account for 96%. VSMCs were observed on the 3 rd day of the culture. The cells showed long shuttle and polygonal morphology. Immunofluorescence technique identified that the expression frequency of SM22-α and α-actin was about 95%. Endothelial exosomes obtained by ultracentrifugation displayed Hsp70/TSG101/CD9/CD63 positive phenotype. VSMCs started to uptake endothelial exosomes after 30 minutes co-culture showed by high-content cell imaging system. After 4 hours,the numbers of endothelial exosomes uptake by VSMCs increased significantly. Cell growth and proliferation curve showed that,at the concentration of 200 μg/ml,the exosomes secreted by HUVEC in which ATG5 decreased could inhibit the proliferation of VSMC. Conclusion ATG5 regulates VSMC proliferation by mediating endothelial exosomes.

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Objective To investigate the effects of liraglutide on glucocorticoid-induce osteoporosis rats and the possible mechanism. Methods Thirty healthy 8-week-old male SD rats were randomly divided into normal control group, dexamethasone group, and dexamethasone + liraglutide intervention group. After 12 weeks of intervention, BMD and bone microstructure were measured by Micro-CT. The kits were used to detect the content of reactive oxygen species(ROS), superoxide dismutase(SOD) and malondialdehyde(MDA) in bone tissue, Western blot was used to detect autophagy specific protein Beclin-1, Atg5, p62/SQSTM1 W and Map1-LC3-Ⅱ expression level. Results In the dexamethasone group, the bone density(BMD), trabecular bone volume ratio(BV/TV), trabecular bone volume(Tb.N), trabecular bone thickness(Tb.Th), and bone size of the distal femur Beam spacing(Tb.Sp) and trabecular bone connection density(Conn.D) were lower than those in the control group. The content of oxidative stress products ROS and MDA in bone tissue increased, the antioxidant enzyme SOD activity was reduced, and the expression levels of autophagy related proteins Beclin-1, Atg5 and Map1-LC3-Ⅱ increased, and the expression level of p62/SQSTM1 W protein decreased(allP<0.05). Bone density and bone microstructure of rats in the liraglutide intervention group were improved, ROS and MDA content in bone tissue reduced, antioxidant enzyme SOD activity was increased, and the expression levels of autophagy-related proteins Beclin-1, Atg5, and Map1-LC3-Ⅱ decreased, and the expression level of p62/SQSTM1 W protein increased(allP<0.05). Conclution Liraglutide can improve osteoporosis caused by glucocorticoids, and its mechanism may be related to adjusting autophagy and improving oxidative stress.

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Objective To investigate the WNT ligands regulated by TCF7 L2 in gastric cancer cells and their functions on the proliferation and metastasis of gastric cancer cells. Methods qPCR and PCR array methods were used to detect the expression of TCF7 L2 and WNT7 b genes in gastric cancer cell lines and clinical specimens. 200 ng/ml recombinant human WNT7 b protein was used to treat gastric cancer cell lines SGC7901 and N87. qPCR, Western blot and dual-luciferase reporter gene detection system were used to detect TCF7 L2 and β-catenin/TCF binding activity, respectively. 2 μg/ml actinomycin D treatment was used to analyze the effect of WNT7 b on TCF7 L2 mRNA stability in N87 cells. CCK-8 kit, wound healing and transwell methods were used to analyze the abilities of proliferation and migration of gastric cancer cells. Results By analyzing PCR array data of SGC7901/TCF7 L2, N87/shTCF7 L2 and their corresponding control cell lines, it was found that WNT7 b was the most remarkable in the influenced WNT ligands by TCF7 L2 expression. The detection of 17 gastric cancer specimens showed that the transcription levels of TCF7 L2 and WNT7 b in gastric cancer tissues were both higher than those in normal adjacent tissues, and there should be a positive correlation between the expressions of TCF7 L2 and WNT7 b. TCF7 L2 transcription levels increased after the 24 h treatment of WNT7 b protein on N87 cells(P<0.001). Meanwhile, TCF7 L2 mRNA stability was enhanced(P<0.05), and TCF7 L2 protein levels and the β-catenin/TCF binding activity were increased(P<0.05). Moreover, it was demonstrated that WNT7 b and TCF7 L2 enhanced the proliferation and migration abilities of gastric cancer cells(P<0.05) through the cytology experiments. Conclution The expression levels of TCF7 L2 and WNT7 b in gastric cancer tissues are higher than those in normal control. There is a mutual expression enhancement between WNT7 b and TCF7 L2, which plays a role in regulating the proliferation and migration of gastric cancer cells together.

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Objective To investigate the regulatory effect of YAP gene on the expression of TGF-α and EGFR in cervical cancer cells infected with HPV. Methods Immunohistochemistry and Western blot were used to detect the expression of YAP, TGF-α and EGFR in HPV-16(+) Ca Ski, HPV-18(+) C4-1 human cervical cancer cells and HPV(-) C33 a human cervical cancer cells. After intervention with the YAP inhibitor Vertepofin, it affected the proliferation of Ca Ski and C4-1 cells and C33 a,as well as the expression of mRNA and protein in YAP,TGF-α,and EGFR. Results The expressions of YAP,TGF-α and EGFR in HPV-16( +) Ca Ski and HPV-18( +)C4-1 human cervical cancer cells were higher than that of HPV(-) C33 a cells; after Vertepofin intervention,the cell proliferation rate decreased. At the same time that YAP was inhibited,the expression of mRNA and protein in TGF-α and EGFR also appeared to be inhibited. Conclusion The Hippo/YAP signaling pathway for EGFR and TGF-α expression in HPV-infected cervical cancer cells can play a positive regulatory role.

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Objective To explore the specific mechanism of LncRNA NRON affecting NFAT activity and progression of atrial fibrosis. Methods The expression level of NRON in atrial tissue was detected by qRT-PCR. Western blot was used to determine the protein levels of collagen Ⅰ, collagen Ⅲ, NFATc3 and pNFATc3. Immunohistochemistry was performed to observe the expression and distribution of collagen Ⅰ in atrial tissue. Atrial fibroblasts were identified by vimentin/troponin immunofluorescence staining. Then the proliferation of fibroblasts was detected by CCK-8 analysis. HE staining was used to observe the morphological changes of cardiac tissue, and finally the myocardial fibrosis was detected by Masson staining. Results NRON inhibited fibroblast proliferation, and the expression of collagen Ⅰ and collagen Ⅲ, activated NFATc3 and nuclide entry. Overexpression of NRON inhibited the cardiac fibrosis induced by NFAT in mice. Conclution The data from this study suggest that NRON alleviates atrial fibrosis by promoting NFATc3 phosphorylation.

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Objective To investigate the effect of miR-664 b-5 p on the biological behavior of esophageal cancer cells EC9706 and TE-1. Methods Quantitative real-time PCR(qRT-PCR) was used to determine the expression of miR-664 b-5 p after transfected with miR-664 b-5 p mimic or inhibitor in esophageal cancer cells EC9706 and TE-1; Cell proliferation was assessed by CCK-8 and colony formation; Migration and invasion of esophageal cancer cells were observed by the transwell chamber assay; The expression of apoptosis-related proteins Bax and Bcl-2 were detected by Western blot. Results After the transfection of miR-664 b-5 p mimic, the expression level of miR-664 b-5 p increased in esophageal cancer cells EC9706 and TE-1(P<0.05), while the transfection of miR-664 b-5 p inhibitor was the opposite(P<0.05); The results of cell proliferation assay showed that the ability of the cell proliferation and the number of colony formation decreased in the miR-664 b-5 p mimic group compared with the mimic NC group(P<0.05), and the ability of the cell proliferation and the number of colony formation in the miR-664 b-5 p inhibitor group was higher than the inhibitor NC group(P<0.05); The results of transwell assay showed that the number of migration cells and invasive cells of esophageal cancer in the miR-664 b-5 p mimic group was lower than that of the mimic NC group(P<0.05), while the number of migration cells and invasive cells in the miR-664 b-5 p inhibitor group was higher than that of the inhibitor NC group(P<0.05); The results of Western blot showed that the expression of Bax increased and Bcl-2 reduced in the miR-664 b-5 p mimic group(P<0.05), but the expression of Bax reduced and Bcl-2 increased in the miR-664 b-5 p inhibitor group(P<0.05). Conclusion miR-664 b-5 p can inhibit cell proliferation,migration and invasion,and promote the apoptosis of esophageal cancer cells EC9706 and TE-1, which provides the foundation to further study the molecular mechanism of miR-664 b-5 p in Kazakh patients with esophageal squamous cell carcinoma.

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Objective To study the effect of high fat diet on metabolism and body fat of offspring mice in different periods(pregnancy and lactation) by feeding maternal mice with high fat diet. Methods ICR pregnant mice were divided into control group(C) and high-fat group(H) randomly,after delivery, each group(mentioned above) was subdivided into two groups: C and H. Therefore, four groups were formed: CC group, CH group, HC group and HH group. After weaning on the 21 st day, the offspring mice of all groups were fed with common feed until the 8 th week(end of adolescence). Then the other three groups were fed with high-fat diet until the 15 th week except the CC group. Results The blood glucose and cholesterol of placenta and fetus in group H were higher than those in group C. At the 8 th week, the serum total cholesterol level of HH group was higher than that of CH group. At the 15 th week, the body weight, body fat percentage and insulin level of CH group were higher than those of the control group, and the fasting blood glucose of HH group was higher than that of CC group. Conclusion High fat diet during pregnancy will lead to the disorder of glucose and lipid metabolism in offspring mice, and the offspring are more likely to be obesity and metabolic disorder after being induced by high-fat diet in adulthood if their mother mice are fed with high-fat diet during lactation.

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Objective To describe the status of the chronic diseases and activities of daily living of the elderly in urban and rural communities, and to explore the correlation between them. Methods The elderly people(age older than 60) from urban and rural communities in Lu'an were recruited to be investigated and to be taken a physical examination by multistage stratified cluster sampling. The chronic disease screening scale and Barthel index were used to evaluate chronic diseases and activities of daily living, respectively. Results A total of 1 080 elderly people completed the survey and 983 people were finally included in the analysis after removing those who did not complete the Barthel index rating scale. In the elderly, the prevalence of chronic diseases was 77.2%(759 cases), and the detection rate of daily living dysfunction was 31.7%(312 cases). Compared with the healthy elderly(17.4%), the rate of daily living dysfunction detection among elderly people with chronic diseases(29.2%) was higher. After adjusting for sociodemographic variables, multivariate logistic regression revealed hypertension(OR=1.747, 95%CI: 1.229～2.484), coronary heart disease(OR=1.614, 95%CI: 1.061～2.457), and stroke(OR=4.942, 95%CI: 2.735～8.871) were associated with impaired daily living ability(P<0.05). Compared with the healthy elderly, the elderly with one chronic diseases(OR=1.851, 95%CI: 1.201～2.853), two chronic diseases(OR=2.918, 95%CI: 1.783～4.773), and more than two chronic diseases(OR=5.490, 95%CI: 2.636～11.430) had a higher detection rate of daily living dysfunction. Nonparametric test found that compared with the healthy elderly, patients with hypertension, coronary heart disease, and stroke had different scores in urinary control, toileting, bed and chair transfer, walking on flat ground, and going up and down stairs(P<0.05). Conclusion Chronic diseases are associated with daily living dysfunction in Lu'an urban and rural communities' elderly people. Hypertension, coronary heart disease, stroke, and chronic comorbidities increase the risk of daily living dysfunction, and these diseases cause different functional impairments.

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Objective To develop an instrument for assessing cancer prevention literacy(CPLI) that available to healthy people.Method The item pool was constructed through literature review, patient interview, and expert consultation. We selected 2,400 residents as the survey objects, and the reliability and validity of the scale were evaluated by kronbach coefficient, Spearman correlation analysis and factor analysis. Using ANOVA and t-test, we analyzed the health belief scores of residents. Result There were 63 items in the CPLI,with the overall Cronbach'α coefficient as 0. 874. Spearman correlation coefficient analysis showed that the content validity was good( the correlation coefficients between any construct index and items within the same construct were 0. 146 ～ 0. 772,while the correlation coefficients between the constructed index and items outside the same construct were-0. 206 ～0. 451). Factor analysis showed that there were 16 common factors of characteristic root>1,and the factor loads of corresponding factors were>0. 50 in 61 items( all of them were within the preset five dimensions). The cumulative variance contribution rate was 68. 22%,indicating that the structure validity was good. The average index of health belief model constructs was( 8. 47 ± 0. 81),with the highest index( 9. 08 ± 1. 45) for the perceived severity construct and the lowest index( 6. 43 ± 1. 95) for the perceived risk construct. Differences in the indices by gender,age,and education were statistically significant. Conclusion CPLI has good reliability and validity. Differentiated strategy tailoring to the different literacy status among groups and constructs are required for future intervening and promoting cancer prevention literacy.

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Objective To examine the association between the health literacy(HL) and the levels of serum inflammatory factors in middle school students, and provide evidence for a deeper understanding of the adverse health effects caused by low HL. Methods A total of 911 junior and high school students from a middle school in Shenyang were enrolled by convenient cluster sampling method in December 2017. Fasting venous blood samples were collected from participants and the value of Tumor Necrosis Factor-α(TNF-α) and Interleukin-6(IL-6) in serum were determined by chemiluminescence. The Chinese Adolescent Interactive Health Literacy Questionnaire(CAIHLQ) was applied to estimate the level of HL. Statistical analyses were performed using Kruskal-Wallis test and Spearman correlation test. Results The students were 13.92～16.83(15.41±1.73) years old, among which 479(52.6%) were boys. The scores of HL of students were 106.34±19.06. The levels of TNF-α and IL-6 were represented by the median(P25,P75), TNF-α was 5.63(4.84, 6.64) ng/L, IL-6 was 3.05(2.43, 3.98) ng/L. The scores of HL in all girls, junior school girls and high school girls were negatively correlated with TNF-α level and spearman correlation coefficients were-0.133,-0.201 and-0.121, respectively(P<0.05). The scores of HL in all junior school students and junior school girls were negatively correlated with IL-6 level and spearman correlation coefficients were-0.343 and-0.708, respectively(P<0.05). Conclusion The scores of HL are negatively correlated with TNF-α and IL-6 level in junior school girls.

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Objective To identify risk factors for anastomotic leakage and to create a nomogram for precise prediction of anastomotic leakage after anterior resection for rectal cancer. Methods A case-control study was conducted to retrospectively analyze the clinicopathological data of 326 consecutive patients who underwent sphincter-preserving surgery for rectal cancer. Univariate and multivariable logistic regression analysis were used to determine independent risk factors associated with postoperative anastomotic leakage. A nomogram was constructed based on the selected independent risk factors to predict anastomotic leakage. The accuracy and predictability of the model were evaluated by using a bootstrapped-concordance index and calibration plots. Results Univariate and multivariate analysis identified the following variables as independent risk factors for anastomotic leakage: gender(male)(OR=2.847,95%CI:1.197～6.771), BMI(≥24 kg/m2)(OR=3.790,95%CI:1.812～7.927), M stage(M1)(OR=3.176,95%CI:1.122～8.989) and distance of tumor to anal verge(≤7 cm)(OR=2.992,95%CI:1.407～6.362). We created a nomogram with high discriminative ability(concordance index, 0.755) validate by the Bootstrap internal verification method. The area under the curve value, which evaluated the predictive performance of external validation, was 0.773(95%CI:0.712～0.844). The validation of the model showed that the predicted values and the observed values were basically consistent, indicating that the nomogram prediction model had a good predictive ability. Conclution Male, BMI≥24 kg/m2,M1 stage and distance from anal verge to tumor≤7 cm were independently associated with anastomotic leakage formation. The nomogram prediction model is helpful to predict the probability of AL after sphincter-preserving surgery.

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Objective To identify the long non-coding RNAs(lncRNAs) differentially expressed in metastatic and non-metastatic nasopharyngeal carcinoma(NPC) patetients and to predict their underlying biological functions. Methods High-throughput sequencing technique was used to detect the expression of lncRNAs in serum of 2 patients with non-metastatic NPC and 3 patients with metastatic NPC. The potential biological mechanism of lncRNAs was evaluated and predicted by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis. Results Compared with the sera of patients with non-metastatic NPC, there were 2 high expression and 12 low expression lncRNAs in the metastasis group(P<0.05). Among them, the difference multiple of lncRNA ENST00000610778 with low expression in the metastasis group was larger. The genes encoding proteins around these lncRNAs were enriched in biological processes such as translation, ribosomal large subunit assembly, viral transcription and ribosomal pathway. Conclusion There are many differentially expressed lncRNAs in the sera of patients with metastatic and non-metastatic NPC, which may have specific biological functions and become new targets for the diagnosis and treatment of metastatic NPC.

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Abstrsct Objective To determine the metabolite of intestinal bacteria,D-lactic acid( D-LA),the product of intestinal mucosa,diamine oxidase( DAO) and markers of inflammation in male patients with alcoholic fatty liver disease( AFLD) and male patients with nonalcoholic fatty liver disease( NAFLD),and to investigate the changes of intestinal mechanical barrier function in male patients with fatty liver disease( FLD). Methods A total of 40 male patients with alcoholic fatty liver disease( AFLD group),91 male patients with nonalcoholic fatty liver disease( NAFLD group),and 82 normal people( normal control group) were selected as the object of study. Questionnaires were sent to them,followed by physical examination,liver ultrasound examination and blood eamination.The physical examination report was collected. A self-designed fatty liver questionnaire was used to investigate the general condition of individuals. Venous blood was sampled to test biochemical indexes,intestinal permeability and inflammatory makers of body. Results The body mass index( BMI),alanine aminotransferase( ALT),aspartate aminotransferase( AST),triglycerides( TG),very low-density lipoprotein cholesterol( VLDL-C),and diamine oxidase( DAO) in NAFLD group and AFLD group were all higher than those in the control group. The high-density lipoprotein cholesterol( HDL-C) in NAFLD group and AFLD group was lower than that in the control group. The concentration of serum D-lactic acid( D-LA) in NAFLD group was higher than that in the control group. All these differences above were statistically significant( P<0. 05). Inflammatory makers suggested that the serum level of tumor necrosis factor-α( TNF-α),interleukin-6(IL-6) and C-reactive protein(CRP) in NAFLD group and AFLD group was higher than that in the control group,and the concentration of TNF-α and CRP in AFLD group was higher than that in NAFLD group(P<0. 05). The correlation analysis showed that the DAO in serum was positively correlated with ALT,AST,TC,TG,and VLDL-C(rs>0,P<0.05),the DAO and D-LA in serum were negatively correlated with HDL-C(rs<0,P<0. 05),and the TNF-α,IL-6 and interleukin-1β(IL-1β) in serum were positively correlated with DAO( rs>0,P<0.05). Conclusion The level of DAO and D-LA in serum of NAFLD patients significantly increased,while the DAO in serum also increased in AFLD patients,suggesting that intestinal mechanical barrier function in patients with fatty liver disease was impaired compared with normal population. The increased serum level of TNF-α,IL-6 and CRP in patients with NAFLD and AFLD,and the level of TNF-α and CRP in AFLD patients was higher than that in NAFLD patients,suggesting that the inflammatory level in fatty liver disease was higher,and the inflammatory level in AFLD patients might be higher.

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Objective To investigate the effect of high-precision transcranial direct current stimulation(HD-tDCS) on cognitive and social functions in convalescent schizophrenics. Methods 100 cases of convalescent schizophrenics admitted to our hospital from January 2018 to June 2019 were selected as the research objects. According to the random number table method, they were divided into two groups, and 50 cases in each group. Both groups used HD-tDCS to stimulate the left dorsolateral prefrontal lobe on the basis of conventional psychiatric treatment. The real stimulation group used 1.5 mA current stimulation for 20 min, and the false stimulation group only used 30 s passing at the beginning and end of stimulation, both once a day, lasting for 10 days. The social function and cognitive function of the two groups were assessed by the inpatient psychiatric social function assessment scale(SSPI) and the Chinese version of the matrix cognitive battery(MCCB) before and end of the treatment, and the adverse reactions of the two groups in HD-tDCS treatment were compared. Results There was no significant difference in the total score of SSPI and MCCB between the two groups before and after treatment(P>0.05); After treatment, the total score of SSPI and all factors in the two groups were higher than those before treatment(P<0.05), and the total score of SSPI and all factors in the real stimulation group were higher than those in the false stimulation group(P<0.05); After treatment, the total score of MCCB, information processing speed, attention/alarm in the real stimulation group were higher than those in the real stimulation group(P<0.05) The scores of perception, working memory, reasoning and problem solving were higher than those before treatment(P<0.05). The patients in both groups were well tolerated to electrical stimulation or false stimulation, and there was no serious adverse event and no significant difference between the two groups(P<0.05). Conclusion HD-tDCS treatment for convalescent schizophrenics is beneficial to promote cognitive rehabilitation and further improve social function, which has a good application prospect.

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Objective To analyze the risk factors influencing postoperative survival of patients with resected gastric adenocarcinoma and stablish a prognostic Nomogram model. Methods The clinicopathological and survival data of 4787 patients with resected gastric adenocarcinoma were collected from the National Cancer Institute′s Surveillance,Epidemiology and End-results(SEER) database.ROC curve was used to determine the optimal cut-off point of lymph node ratio(LNR).The Kaplan-Meier method was used to calculate the cumulative survival rate,and the significant difference was evaluated by the Log-rank test.The Cox multivariate regression analysis was applied to evaluate the independent prognostic factors,and a prognostic Nomogram was established by these independent prognostic factors.Then the Harrell′s C-index and decision curve analysis(DCA)were used to estimate the accuracy and clinical utility of the Nomogram system. Results The optimal cutoff value of LNR was 0.075.Univariate and multivariate analysis showed that LNR,AJCC 7 th T stage, N stage, M stage were independent prognostic factors of patients with resected gastric adenocarcinoma(P<0.05). Using these factors to build the Nomogram prognosis model, the internal verification C-index was 0.708(95%CI:0.696～0.720) and the consistency of calibration curves was rather good. The Nomogram system showed good accuracy in predicting OS of 1-year,3-year. DCA also showed that the Nomogram prediction model had higher clinical benefit than the AJCC 7 th TNM system. Conclusion Age, LNR, AJCC 7 th T stage,N stage,and M stage are independent prognostic factors of patients with resected gastric adenocarcinoma(P<0.05).The Nomogram provided improved discrimination and higher clinical benefit than the AJCC 7 th TNM system.

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Objective To sequence the T cell receptor(TCR) of patients with Graves ophthalmopathy(TAO) before and after the treatment of intravenous injection of methylprednisolone, and to further explore the immunologic mechanism of the treatment of Tao by intravenous injection of methylprednisolone, so as to provide theoretical basis for the new immunologic treatment. Methods Six patients with TAO were collected from the outpatient department of the First Affiliated Hospital of Xiʼan Jiaotong University. The peripheral blood was collected at three time points of diagnosis, the end of the first shock treatment and the end of the third shock treatment. The peripheral blood mononuclear cells( PBMCs) were extracted and RNA was extracted for TCR immunohistochemistry library sequencing,followed by high-throughput sequencing data mining analysis. Results After three hormone shocks,there was no significant difference in TCR diversity index,VJ family migration and VJ family frequency among the improved TAO patients. Conversely,the high frequency VJ family showed clonal expansion in worsening TAO patients. TAO remission was linked with the decrease of Th17 cells. Conclusion The mechanism of intravenous shock therapy of TAO by methylprednisolone is the reestablishment of immune balance rather than the elimination of autoreactive cloning.

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To explore the difference of expression profiles of microRNA(miRNA) in the exosomes of endometrial stromal cells(ESC) from different sources. The exosomes were isolated from 40 patients with endometriosis(EMs)(study group) and 40 patients with cervical intraepithelial neoplasia(CIN) Ⅲ underwent uterine surgery(control group) after ESC primary culture. Chip was used to detect the difference of miRNA expression profile between the two groups. Real-time fluorescence quantitative PCR(qRT-PCR) was used to detect the levels of miR-205 and miR-542-3 p in ESC exosomes. The target genes related to EMs of miRNA with higher up-regulated and down-regulated multiples were predicted. There were significant differences in the expression of 39 kinds of miRNA in ESC exosomes between the study group and the control group(P<0.05), among them, there were 14 up-regulated miRNA, with the up-regulated multiple of miR-205 was the highest; there were 25 down-regulated miRNA, with the down-regulated multiple of miR-542-3 p was the highest. qRT-PCR showed that there was no significant difference between the levels of miR-205 and miR-542-3 p in ESC exosomes with the chip detection multiples(P>0.05). 26 genes related to EMs, such as PTEN, FBN1 and S100 A2, were predicted to be differentially expressed target genes of miRNA. There are many differentially expressed miRNA in ESC exosomes of EMs, targeting a variety of EMs-related genes.

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Established Fe3O4@ZIF-67 magnetic dispersive micro-solid phase extraction(DMSPE) combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS) to determine 6 penicillins(PCs) in water samples residual method. Under the optimal extraction conditions, the detection limit of 6 PCs in water samples was 0.01～0.06 μg/L, and the limit of quantification was 0.03～0.19 μg/L; under the three concentration levels of 0.1, 1.0, 10.0 μg/L, the recovery rates of the 6 PCs in river water and aquaculture water samples were 79.2%～96.6% and 75.9%～94.8%, respectively; the matrix effect of the six PCs in river water and aquaculture water were 90.3%～102.2% and 89.6%～105.6%. Fe3O4@ZIF-67 has the ability to efficiently adsorb PCs. Combined with LC-MS/MS, it can realize the simultaneous rapid detection of 6 PCs in water samples.

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All of 231 patients included 293 zirconia or metal-based single crowns and 76 FDPs were evaluated. The estimate cumulative success rates of zirconia ceramic group and metal ceramic group were 79.61% and 84.74% respectively, and the estimate cumulative survival rates were 90.29% and 91.58%. There was no significant difference between the two groups in terms of success and survival rate. The chipping rate in zirconia ceramic group was 12.62% and 5.79% in metal ceramic group, there was significant difference between the two groups. The success and survival rates for FDPs were 75.00% and 88.16% respectively.