Abstract:The homeostasis of gastrointestinal microecology plays a critical role in maintaining normal host physiological activity. He－licobacter pylori（Hp） is a common pathogenic bacterium in the stomach and can cause gastric mucosal injury and even gastric cancer after infection. Hp colonization in the stomach can affect the homeostasis of gastrointestinal microecology，and gastrointestinal micro－biota may be involved in the pathogenic process of Hp and thus affect the outcome of Hp infection. This article reviews the interaction between Hp infection and gastrointestinal microbiota and its influence on the pathogenesis of Hp，and reveals the influence of Hp in－fection on gastrointestinal microecology，the role of gastrointestinal microecology in the pathogenesis of Hp infection，and the signifi－cance of probiotics in Hp eradication.

Abstract:Objective：To examine the effects of Typhonium giganteum extract（TGE） on the proliferation and metastasis of human gastric carcinoma cell line GC9811 and to elucidate its possible mechanism of action. Methods：TGE was isolated and purified by phyto－chemistry，and the inhibitory effect of different concentrations of TGE on GC9811 cell proliferation at various time points was mea－sured by MTT colorimetric assay. The effect of TGE on cell migration was determine by cell scratch assay and Transwell migration assay. The mRNA and protein expression of matrix metalloproteinase-9（MMP-9），matrix metalloproteinase-2（MMP-2），and E-cad－herin was measured by real-time quantitative PCR and Western blot，respectively. Results：The MTT assay showed that TGE significantly inhibited the growth of GC9811 cells in a dose-dependent manner. The cell scratch and Transwell migration assays demonstrated that the migratory ability of GC9811 cells decreased as TGE concentration increased. Furthermore，MMP-2 and MMP-9 expression was significantly downregulated and E-cadherin expression was significantly upregulated in the 30 g/L TGE group than in the 15 g/L TGE group. Conclusion：TGE can significantly inhibit the proliferation and metastasis of GC9811 cells，and its mechanism of action may be related to the upregulation of E-cadherin expression and downregulation of MMP-9 and MMP-2 expression. TGE is a highly potent and minimally toxic antitumor lead compound worthy of further investigation.

Abstract:Objective：To screen out the key circRNAs that regulate the proliferation and invasion of adenocarcinoma of the esophagogastric junction（AEG） by high-throughput RNA sequencing and bioinformatics analysis，and to preliminarily explore their modes of action. Methods：RNAs were extracted from 22 pairs of adenocarcinoma of the esophagogastric junction and adjacent normal mucosa tissues. Differentially expressed genes were screened out by high-throughput RNA sequencing（sequencing depth 200×） and bioinformatics cloud platform. Meanwhile，a co-expression network and an endogenous RNA competition mechanism（ceRNA） network among circRNAs-miRNAs-mRNAs were constructed for differentially expressed genes. The construction of co-expression network of RNA maps and the negative correlation analysis of endogenous competitive RNAs were used to screen for circRNAs that may play a key role in the development of AEG，and to predict the expression of miRNAs and mRNAs which have ceRNA relationship with the circRNA. qRT-PCR and other methods were used to verify the expression of circRNA in gastric cancer tissues and adjacent tissues. Results：A total of 46553 circRNAs were predicted by high-throughput RNA sequencing，and 26 differentially expressed circRNAs were obtained after strict quality control of sequencing data（fold change ≥2 or fold change ≤0.5，false discovery rate<0.05）. Among them，10 circRNAs were up-regulated and 16 circRNAs were down-regulated. Tissue samples were analyzed by mRNA and miRNA sequencing，and the ceRNA network among circRNAs-miRNAs-mRNAs，co-expression network of RNA maps，and endogenous competitive RNA negative correlation analysis were constructed. The results showed that has_circ_0007766 was highly specifically expressed in AEG tumors，which may play a key role in the development and progression of AEG. Meanwhile，it was also found that miR4492 had binding sites with both circRNA0007766 and MET mRNA. Conclusion：The has_circ_0007766 may regulate the proliferation and invasion of adenocarcinoma of the esophagogastric junction through the ceRNA network of mir4492- and circRNA0007766-MET mRNA，which may be the key circRNA for the development and progression of AEG.

Abstract:Objective：To investigate the effects of long non-coding RNA MALAT1 on the proliferation，migration，and invasion of gas－tric cancer through regulating the miR-22/snail axis. Methods：The expression of MALAT1 was inhibited by siRNA technology，and miR-22 and snail were overexpressed；real-time PCR was used to determine the expression levels of MALAT1，miR-22，and snail in gastric cancer cell lines（n=3）；Western blot was used to determine the expression level of snail protein in NC group and si-MALAT1 group（n=3）；CCK8 assay was used to determine the cell absorbance values in NC group，si-MALAT1 group，miR-22 group，miR-22+snail group，and si-MALAT1+miR-22 group at 12 h，24 h，48 h，and 72 h（n=3）；wound healing assay was used to determine the mi－gration rate in NC group，si-MALAT1 group，and miR-22 group（n= 3）；transwell assay was used to determine the number of trans－membrane cells in NC group，si-MALAT1 group，miR-22 group，miR-22+snail group，and si-MALAT1+miR-22 group（n=3）；dual luciferase assay was used to determine the fluorescence activity in NC+pMIR-MALAT1-WT group，NC+pMIR-MALAT1-MUT group，miR-22+pMIR-MALAT1-WT group，miR-22+pMIR-MALAT1-MUT group，NC+pMIR-Snail-WT group，NC+pMIR-Snail-MUT group，miR-22+pMIR-Snail-WT group，and miR-22+pMIR-Snail-MUT group（n=3）. The t-test was used for comparison between two independent samples；one-way analysis of variance was used for comparison between multiple sets of data，and repeated measures analysis of variance was used for time-associated fac－tors. Results：The expression level of MALAT1 in gastric cancer cell lines was significantly higher than that in normal gastric mucosal epithelial cells（all P=0.000）；after underexpression of MALAT1，AGS and SGC-7901 had significantly decreased prolifer－ative ability，migration ability，and invasion ability（P1=0.001，0.018，and 0.024，respectively；P2=0.005，0.007，and 0.015，respectively）；meanwhile，the dual luciferase assay results showed that MALAT1 could targetedly bind to miR-22，and miR-22 could targetedly bind to snail（both P=0.001）. The expression level of miR-22 in gastric cancer cells was significantly lower than that in normal gastric mucosal epithelial cells（P<0.05）；after overexpression of miR-22，AGS and SGC-7901 had significantly decreased proliferative ability，migration ability，and invasive ability（P1=0.004，0.034，and 0.037，respectively；P2=0.009，0.005，and 0.011，respectively）. Concomi－tant underexpression of MALAT1 and overexpression of miR-22 could further enhance the inhibitory effect on the proliferation and invasion of AGS and SGC-7901 after underexpression of MALAT1（P1=0.022 and 0.024，respectively；P2=0.006 and 0.015，respectively）；concomitant overexpression of miR-22 and snail could reverse the inhibitory effect of miR-22 on the proliferation and invasion of AGS and SGC-7901（P1=0.003，and 0.015，respectively；P2=0.004 and 0.01，respectively）. Conclusion：There is significantly overex－pressed MALAT1 in gastric cancer cells，which can facilitate the proliferation，migration，and invasion of gastric cancer by regulating the miR-22/snail axis.

Abstract:Objective：To study the regulation and control of the phenotype and function of natural killer（NK） cells by the primary cells of esophageal squamous cell carcinoma（ESCC）. Methods：Fresh ESCC tissue was collected，from which primary ESCC cells were cultured in vitro by the modified tissue block enzymolytic method，and identified using a subcutaneous tumor-formation model in nude mice and the immunohistochemical technique. Human peripheral blood mononuclear cells were collected from healthy subjects，from which the NK cells were isolated by negative-selection magnetic beads，and co-cultured with the supernatant of the primary ESCC cells obtained above. The cultured cells were divided into groups of NC-NK，ESCC1-NK，and ESCC2-NK according to their respective culture conditions. Flow cytometry was used to determine the molecular expression both on the surface of and within the NK cells. The subcutaneous tumor-formation model and lung metastasis model were used to study the effect of primary ESCC cells on the killing effect of NK cells in nude mice. Results：Compared with the NC-NK group，the ESCC1-NK group and the ESCC2-NK group had significantly reduced expression of the activated receptors NKG2D（56.84±1.28 vs. 41.89±2.17 and 29.40±1.32；P=0.008 and 0.001，respectively），NKP30（45.79±1.90 vs. 19.54±1.27 and 26.59±1.47；P=0.001 and 0.003，respectively），and CD16（84.82±1.38 vs. 55.95±2.29 and 36.07±1.97；P=0.001 and 0.000，respectively） as well as significantly reduced expression of the effector molecule granzyme B（94.87±1.04 vs. 80.52±0.99 and 78.67±2.73；P=0.013 and 0.008，respectively） and the prolif－eration-associated antigen Ki67（70.15±2.35 vs. 52.31±1.74 and 50.02±2.68；P=0.017 and 0.011，respectively） in NK cells. There were no significant differences between the three groups in the expression of the activated receptors NKP46，CD226，and NKP44 as well as perforin and the inhibitory receptors NKG2A and CD158b. Animal experiments showed that there was a significant difference between the ESCC1-NK group and the NC-NK group in the volume of subcutaneous tumors（F=54.689，P=0.000） with a significantly greater number of lung metastases observed in the ESCC1-NK group（41.00±3.11 vs. 25.25±2.32，t=4.068，P=0.007）. Conclusion：Primary ESCC cells may suppress the tumor-killing effect of NK cells by inhibiting the activation and proliferation of NK cells and down-regulating the expression of the effector molecules in NK cells，thereby escaping the immune surveillance in the body.

Abstract:Objective：To investigate the interaction between the polymorphisms of hepatitis B virus（HBV）-related CD8+ T cell in－hibitory genes and HBV mutations and its influence on the outcome of HBV infection. Methods：A total of 239 healthy controls，429 patients with spontaneous clearance，and 858 patients with chronic HBV infection were enrolled. A total of 28 tagging single nucleotide polymorphisms were selected from 5 CD8+ T cell inhibitory genes for genotyping. Direct sequencing was performed to determine HBV mutations. Results：The interactions between the polymorphisms of CD8+ T cell inhibitory genes（rs485618，rs4656942，and rs3766377 in CD244 and rs6746608 in BIM） and HBV mutations（T53C，A166C，T216C，A293G，A1762T，G1764A，and A1762T/G1764A） had a great influence on the risk of liver cirrhosis（LC） or hepatocellular carcinoma（HCC）. Similar results were obtained by multifactor dimensionality reduction analysis. Conclusion：The polymorphisms of CD244 and BIM genes might be involved in the regulation of immune selection of LC- or HCC-related HBV mutations，and the interactions between them may affect the progression of chronic HBV infection.

Abstract:Objective：To investigate the effect of ginsenoside Rg1 on non-alcoholic fatty liver disease（NAFLD） in mice induced by high fat diet and its possible mechanism. Methods：A total of 42 healthy female adult C57/BL mice were randomly divided into the control group，model group，Rg1 low dose group，Rg1 high dose group，and metformin group，with 9 mice in each of the previous two groups and 8 in each of the latter. The control group was fed with the normal diet，while the others were fed with the high fat diet，and the feeding was continued for 16 weeks. Then the mice were treated with gastric perfusion，among them，the control group and the model group were treated with normal saline 20 mL（kg·d），Rg1 low dose group with Rg1 20 mg/（kg·d），Rg1 high dose group with Rg1 40 mg/（kg·d），and metformin group with Rg1 150 mg/（kg·d）. After 4 weeks of treatment，the serum and liver tissues of each group were collected. The pathological morphology of liver tissue was observed. The serum transaminase，lipid levels，the content of malondialdehyde（MDA），superoxide dismutase（SOD），free fatty acids（FFA） and the expression of endoplasmic reticulum stress（ERS） related proteins and inflammasome were measured. Results：In Rg1 low dose group and high dose group，the levels of serum alanine aminotransferase（ALT） were （41.87±10.64） and（43.46±13.84） U/L，the levels of aspartate aminotransferase（AST） were （159.56±21.29） and （159.56±25.01） U/L，and the levels of triglycerides（TG） were （0.69±0.15） and （0.75±0.12） U/L respectively，all of which were significantly lower than those in the model group（P=0.001，P=0.010，P=0.000）. And the fatty degeneration degree of liver tissue in the two groups was also significantly smaller than that in the model group（P=0.000）. Rg1 could reduce the contents of FFA and MDA，increase the vitality of SOD（all P=0.000），and down-regulate the expressions of GRP78，CHOP，Caspase12，NLRP3 and IL-1β（P=0.000，P=0.003）. Rg1 can also reduce the expres－sion of endoplasmic reticulum stress related proteins and in－flammasome activation（P=0.000，P=0.003）. Conclusion：Rg1 may improve NAFLD by improving the activity of antioxidant en－zymes and inhibiting the endoplasmic reticulum stress and in－flammasome activation.

Abstract:Objective：To investigate the effect of hepatitis B virus（HBV） replication on the expression of inhibitor of differentiation （Id） family in hepatocytes. Methods：The methods of qRT-PCR and Western blot were used to measure the expression of Id family in hepatoma cells with transient or stable HBV replication，and the results were further validated in normal liver cells transfected with PCH9-HBV1.1 HBV replicating plasmid and HBV transgenic mouse liver tissue cells. HepG2 cells were transfected with plasmids with overexpression of hepatitis B virus S protein（HBs），hepatitis B virus core protein（HBc），hepatitis B virus DNA polymerase（HBp），or hepatitis B virus X protein（HBx），the change in the expression of Id family was measured in these groups and the vector control group，and the regulatory effect of four HBV-en－coded proteins on Id family was analyzed and compared. Lu－ciferase reporter gene assay was used to analyze the effect of virus component proteins on the activity of Id promoter. Results：Com－pared with the control group，the group of HepG2 cells with transient transfection of HBV plasmids had significant reductions in the mRNA and protein expression of Id1 and Id3（mRNA：t=3.952 and 3.189，P=0.017 and 0.033；protein：t=10.532 and 4.155，P=0.000 and 0.014）；compared with the HepG2 control group，the HepG2.2.15 group had significantly lower mRNA and protein expression of Id1 and Id3（mRNA：t=5.553 and 7.211，P=0.005 and 0.002；protein：t=4.193 and 3.849，P=0.014 and 0.018）；compared with the HepAD38（Tet+） group，the HepAD38（Tet-） group had significantly lower protein expression of Id1 and Id3（t=3.052 and 3.712，P=0.038 and 0.021）. In addition，HBV replication significantly inhibited the mRNA and protein expression of Id1 and Id3 in LO2 cells and mouse liver tissue（mRNA：t=14.564，3.281，3.489，and 3.495，P=0.000，0.030，0.025，and 0.025；protein：t=5.651，5.336，4.948，and 5.149，P=0.005，0.006，0.008，and 0.007）. Among the HepG2 cells transfected with HBV-encoded protein plasmids，the HBc group had the greatest reductions in the mRNA expression of Id1 and Id3 compared with the vector control group（77.7% and 76.2%，F=9.945 and 37.528，t=6.481 and 10.915，P=0.000 and 0.000），and Western blot showed that the HBx group had the greatest reductions in the protein expression of Id1 and Id3（86.2% and 68.4%，F=38.225 and 7.159，t=12.550 and 5.295，P=0.000 and 0.001）. The dual-luciferase reporter gene assay showed that the HBc group had the greatest reduction in the promoter activity of Id1 and Id3（62.2% and 56.3%，F=16.530 and 5.210，t=5.442 and 3.222，P=0.000 and 0.019）. Conclusion：HBV can inhibit the expression of Id1 and Id3 in liver tissue and hepatocytes. HBc has the most significant effect in downregulating the mRNA expression of Id1 and Id3，while HBx has the most significant inhibitory effect on the protein expression of Id1 and Id3.

Abstract:Objective：To construct a recombinant eukaryotic expression vector of human matrix metalloproteinase 2（Mmp-2） and in－vestigate the effect and mechanism of action of MMP-2 on the migration and invasion of hepatocellular carcinoma cells. Methods：The human hepatocellular carcinoma SMMC-7721 cells were used as the research model，and the recombinant eukaryotic expression vector pEYFP-Mmp-2 was constructed by gene recombination technology. The cells of pEYFP-Mmp-2 transient transfection model overexpressed MMP-2-YFP，while the cells of siRNA transfection silencing model expressed endogenous MMP-2. Then the above cells were divided into five groups，namely control group，MMP-2 silencing control group（siCON），MMP-2 silencing group（siMMP-2），overexpressed MMP-2 fusion protein control group（ovNC），and overexpressed MMP-2 fusion protein group（ovMMP-2）. Wound healing assay was used to determine cell migration，and Transwell chamber assay was used to evaluate cell invasion. Calpeptin，a specific inhibitor of calcium-activated neutral pro－tease（calpain），inhibited the activity of calpain. The expression levels of MMP-2，MMP-2-YFP，E-cadherin，N-cadherin，and vimentin were measured by Western blotting. Results：The human Mmp-2 recombinant eukaryotic expression vector pEYFP-Mmp-2 was successfully constructed. High expression of MMP-2-YFP was observed in the ovMMP-2 group transfected with the pEYFP-Mmp-2. Compared with the siCON group，the siMMP-2 group showed a significant reduction in the expression level of endogenous MMP-2（P=0.000）. Meanwhile，the cell migration in the siMMP-2 group decreased significantly at 12 h，24 h，and 48 h（P=0.000 or P=0.001），and the cell invasion also decreased significantly at 48 h（P=0.004）. Furthermore，a significant up-regulation of E-cadherin and significant down-regulations of both N-cadherin and vimentin were manifested in siMMP-2 cells（P=0.000）. Compared with those in the ovNC group，the cell migration in the ovMMP-2 group increased significantly at 12 h，24 h，and 48 h（P=0.015 or P=0.001），and the cell invasion increased significantly at 48 h as well（P=0.011）. In addition to those，the ovMMP-2 group also had a significant down-regulation of E-cadherin along with significat up-regulations of N-cadherin and vimentin（P=0.003 or P=0.001）. Results also demonstrated that calpeptin pretreatment significantly reduced the cell migration and invasion，up-regulated the expression level of E-cadherin，and down-regulated the expression levels of N-cadherin and vimentin in the ovMMP-2 group（P=0.000）. Conclusion：In this study，the recombinant eukaryotic expression vector pEYFP-Mmp-2 is successfully constructed by gene recombination technology，which successfully expresses the MMP-2-YFP fusion protein. It finds that MMP-2 mediates the migration，invasion，and epithelial-mesenchymal transition of hepatocellular carcinoma SMMC-7721 cells by regulating calpain.

Abstract:Objective：To investigate the expression and clinical significance of the KIF2C gene in hepatocellular carcinoma by data mining. Methods：Oncomine and GEPIA databases were used to analyze the expression of the KIF2C gene in hepatocellular carcinoma. GEPIA database was used to investigate the correlation of KIF2C expression with pathological classification and survival time. STRING database was used to determine the proteins interacting with KIF2C. Results：The analysis using Oncomine and GEPIA databases showed high mRNA expression of KIF2C in hepatocellular carcinoma tissue（P=0.000），and its expression level was nega－tively correlated with the prognosis of hepatocellular carcinoma（P=0.000）；the expression level of KIF2C increased with the increase in pathological classification. The analysis using STRING database showed that PLK1，AURKB，and CENPA proteins were associated with KIF2C. Conclusion：Tumor gene database mining shows that KIF2C is highly expressed in hepatocellular carcinoma tissue and is associated with patient prognosis，which provides a theoretical basis for research on the pathogenesis of hepatocellular carcinoma and anti-tumor targeted therapy.

Abstract:Objective：To explore the application value of real-time shear wave elastography（SWE） in diagnosing and staging of nonal－coholic fatty liver disease（NAFLD）. Methods：A total of 135 patients diagnosed with nonalcoholic fatty liver disease pathologically or operatively were enrolled to undergo SWE before surgery，and the means of Young’s modulus（Mean） were acquired. The correlation between stages of NAFLD and the Means was analyzed，and the Means of patients at different stages were compared. Finally the receiver operating characteristic（ROC） curve was conducted to evaluate the efficiency of Mean for diagnosing NAFLD at all stages. Results：There were significant differences among Means of NAFLD at different stages（P<0.05），and the Spearman correlation coeffi－cience between Mean and the stage of NAFLD was 0.916（P<0.05）. The area under ROC curve（AUC） of nonalcoholic steatohep－atitis was 0.971（95%CI=0.949 to 0.994），with the sensitivity of 91.3%，the specificity 97.0%，and the threshold value 7.30 kPa. The AUC of fibrosis was 0.984（95%CI=0.969 to 0.999），with the sensitivity of 98.6%，the specificity 88.7%，and the threshold value 9.05 kPa. And the AUC of cirrhosis was 0.965（95%CI=0.940 to 0.990），with the sensitivity of 90.0%，the specificity 88.5%，and the threshold value 11.35 kPa. Conclusion：The Mean of real-time SWE can reflect the severity of NAFLD and is a sensitive indi－cator for NAFLD stages. Consequently it is beneficial to apply SWE in clinical diagnosis and treatment for NAFLD.

Abstract:Objective：To measure the frequency of myeloid-derived suppressor cells（MDSCs） in peripheral blood and serum S100A9 level in patients with colorectal cancer（CRC），to analyze the correlation between the two indices，and to investigate their potential application in the clinical diagnosis and treatment. Methods：A total of 82 clinical peripheral blood samples （including 52 samples from patients with CRC and 30 samples from healthy subjects[as controls]） and related electronic medical records were collected. The frequency of MDSCs in peripheral blood mononuclear cells（PBMCs） was measured by flow cytometry. The serum S100A9 level was measured by a double antibody sandwich enzyme-linked immunosorbent assay. SPSS and Graphpad Prism were used for the Mann-Whitney test，Spearman R analysis，and receiver operating characteristic（ROC） curve analysis. Results：Compared with the controls，the patients with CRC had significantly increased frequency of MDSCs in PBMCs and serum S100A9 level（P<0.01），with a positive correlation between MDSC frequency and serum S100A9 level（r=0.38，P=0.01）. Compared with the patients who were at Dukes’ stage A/B and without any lymph node metastasis，the patients who were at Dukes’ stage C/D and with lymph node metastasis had significantly increased MDSC frequency and serum S100A9 level（P<0.01）；meanwhile，the area under the ROC curve was greater than 0.7 for both of the above indices（P<0.01），indicat－ing that both indices exhibited a favorable value in the diagnosis of CRC and the evaluation of the stage and metastasis of CRC. Conclusion：Patients with CRC have a significantly increased frequency of MDSCs in PBMCs and serum S100A9 level；therefore，both indices may be potential biomarkers that can contribute to the staging of CRC and predict the metastasis of CRC.

Abstract:Objective：To investigate the value of 18F-fluorodeoxyglucose（18F-FDG） PET/CT combined with common serum pancreatic tumor markers in the diagnosis of recurrence and metastasis after surgery for early resectable pancreatic cancer. Methods：A retro－spective analysis was performed for the clinical data of 53 patients who were suspected of recurrence and metastasis after surgery for early resectable pancreatic cancer and underwent PET/CT，in order to explore the value of PET/CT combined with pancreatic tumor markers in the diagnosis of recurrence and metastasis after surgery for early resectable pancreatic cancer. Results：The combination group[PET/CT+carbohydrate antigen 19-9（CA19-9）+carbohydrate antigen 242（CA242）+carbohydrate antigen 50（CA50）] had a sen－sitivity of 97.83%，a specificity of 85.71%，an accuracy of 96.23%，a positive predictive value of 97.83%，and a negative predictive value of 85.71% in the diagnosis of recurrence and metastasis after surgery for early resectable pancreatic cancer. The combination group had a significantly higher sensitivity than the CA19-9 group，the CA242 group，and the CA50 group（P<0.05）. The combination group had significantly higher accuracy than the PET/CT group，the CA19-9 group，the CA242 group，and the CA50 group（P<0.05）. Serum CA19-9 level was positively correlated with maximum standardized uptake value of PET/CT（r=0.583，P=0.018）. Conclusion：18F-FDG PET/CT combined with common serum pancreatic tumor markers has high sensitivity，accuracy，and positive predictive value in the diagnosis of recurrence and metastasis after surgery for early resectable pancreatic cancer. It can identify recurrence and metastasis after pancreatic cancer surgery as early as possible to help patients get timely treatment.

Abstract:Objective：To investigate the effect of donor blood sodium and donor-recipient blood sodium difference on patients after liver transplantation. Methods：A retrospective analysis was performed for the clinical data of 20 donors who underwent donor organ main－tenance and 20 patients who underwent liver transplantation in our hospital from July 2016 to May 2018. Related clinical data were analyzed. Results：There were no significant differences in the highest levels of total bilirubin，alanine aminotransferase（ALT），and aspartate aminotransferase（AST），recovery time of liver function，length of stay in the intensive care unit，and length of hospital stay after liver transplantation between the blood sodium >155 mmol/L donor group and the blood sodium <155 mmol/L donor group. According to donor blood sodium at the time of transplantation，the patients were divided into donor blood sodium >155 mmol/L group and donor blood sodium <155 mmol/L group，and the highest ALT level after surgery in the donor blood sodium <155 mmol/L group was significantly higher than that in the donor blood sodium >155 mmol/L group（747.17±375.34 U/L vs. 357.00±190.50 U/L，t=-2.700，P=0.015）. According to donor-recipient blood sodium difference（ΔNa），the patients were divided into ΔNa <10 mmol/L，ΔNa=10-20 mmol/L，and ΔNa >20 mmol/L groups，and there was a significant difference in the highest ALT level after liver transplantation between the three groups（805.00±332.90 U/L vs. 329.75±237.43 U/L vs. 329.60±186.21 U/L，F= 6.714，P=0.007）；the highest ALT level after liver transplantation in the ΔNa<10 mmol/L group was significantly higher than that in the other two groups（P=0.012 and 0.007），and there was no significant difference between the ΔNa=10-20 mmol/L group and the ΔNa>20 mmol/L group（P=0.999）. ΔNa was nega－tively correlated with the highest ALT level after liver trans－plantation（r=-0.579，P=0.007），and the highest ALT level after liver transplantation increased with the reduction in ΔNa. The linear regression analysis showed that model for end-stage liver disease（MELD） score was an independent risk factor for infec－tion after liver transplantation. Conclusion：Donor blood sodium <155 mmol/L may result in an increase in the highest ALT level after liver transplantation. The highest ALT level after liver transplantation increases with the reduction in ΔNa. MELD score is an inde－pendent risk factor for infection after liver transplantation.

Abstract:Objective：To construct an efficient reverse genetics system for EV71 virus，and to rapidly obtain the rescued viruses and identify their activity. Methods：A recipient plasmid pHM-ccdB containing human RNA polymerase Ⅰ promoter，ccdB suicide gene，and murine terminator（mTer） was constructed，and the AarⅠ enzyme cut site was introduced into both sides of the ccdB gene. The virus genome was amplified by PCR in two steps to avoid the AarⅠ enzyme cut site contained in the EV71 genome. The necessary AarⅠ enzyme cut site was introduced into the primer；then the products were connected with the target vector by Golden Gate Clone technique to obtain the rescued plasmid of EV71 virus（pHT-EV71）；after the pHT-EV71 was transfected into RD cells，the rescued EV71 viruses were obtained. The progeny viruses were identified by RT-PCR，virus titer，Western blot，and electron microscopy. Results：The pHT-EV71 was successfully constructed by introducing ccdB lethal gene and Golden Gate Clone technique. After the pHT-EV71 was transfected into RD cells，obvious cytopathic effect was observed. The rescued progeny viruses obtained were ampli－fied by RT-PCR with EV71-specific primers，and specific bands of approximately 1 900 bp were observed. The viruses can be bound with EV71 VP1 specific antibody as showed by Western blot. Spherical virus particles of 20 to 30 nm in size were observed by transmission electron microscopy. After eight generations of progeny viruses in RD cells，the virulence of the progeny viruses was stronger than that of the parental viruses（6.35 lgTCID50/mL）. Conclusion：A rapid and efficient method to construct the pHT-EV71 with a 100% efficiency was successfully established through introducing the ccdB gene and Golden Gate Clone tech-nique. This method provides a new strategy for the construction of reverse genetics system for positive-strand RNA viruses and a technical platform for further study on the pathogenesis and vaccine preparation of EV71 virus.

Abstract:Objective：To explore the upstream regulatory molecules of liver kinase B1（LKB1） and to investigate their effect on LKB1. Methods：First，the UbiBrowser database was used to predict the possible upstream regulatory molecules of LKB1，and the Smurf1 gene that may regulate LKB1 was screened out according to the score. Then，the Smurf1- and LKB1-related overexpression plasmids were constructed and transfected into HEK293T cells，and the changes in the expression of LKB1 protein were detected by Western blot. Finally，the possible molecular mechanism of Smurf1 regulating LKB1 was explored by quantitative polymerase chain reaction and co-immunoprecipitation. Results：①The LKB1- and Smurf1-related overexpression plasmids were successfully constructed. ②The overexpression levels of 0，0.5，1，and 2 μg of wild-type Smurf1 in HEK293T cells corresponded to LKB1 protein expression levels of 0.026 2±0.000 7，0.072 7±0.000 3，0.130 1±0.001 3，and 0.431 5±0.001 0，respectively. One-way analysis of variance（ANOVA） showed a result of F=79 636.433 and P=0.000. Further analysis by the least significant difference t（LSD-t） test yielded P values of 0.000，0.000，and 0.000 for the 0.5，1，and 2 μg groups，respec－tively，compared with the 0 μg control group. ③The overexpres－sion levels of 0，0.5，1，and 2 μg of mutant Smurf1 in HEK293T cells corresponded to LKB1 protein expression levels of 0.018 0 ±0.000 1，0.053 0±0.000 4，0.125 0±0.001 0，and 0.200 4±0.001 7，respectively. One-way ANOVA showed a result of F=12 887.993 and P=0.000. Further analysis by the LSD-t test yielded P values of 0.000，0.000，and 0.000 for the 0.5，1，and 2 μg groups，respectively，compared with the 0 μg control group. ④With GAPDH as the internal reference substance，the relative mRNA expression levels of LKB1 in the Smurf1 non-overexpression group and Smurf1 overexpression group were 1.085 1±0.412 5 and 2.398 2±1.766 9，respectively. The P value was 0.257 by independent samples t-test analysis. ⑤With β-actin as the internal reference substance，the relative ubiquitination levels of LKB1 in the Smurf1-untreated group，wild-type Smurf1-treated group，and mutant Smurf1（C699A）-treated group were 1.823 8±0.027 8，0.088 1±0.005 1，and 0.212 7±0.006 2，respectively. One-way ANOVA showed a result of F=6 721.953 and P=0.000. Further analysis by the LSD-t test yielded P values of 0.000 and 0.000 for the wild-type Smurf1-treated group and mutant Smurf1-treated group，respectively，com－pared with the Smurf1-untreated group. Conclusion：These results indicate that Smurf1 is involved in the regulation of the stability of LKB1 protein by reducing its polyubiquitination.

Abstract:Objective：To explore the expression of CD146 in polyethylene glycol（PEG）-induced choroidal neovascularization（CNV） in a mouse model and its significance. Methods：Sixty 8-week-old male C57BL/6J mice were randomly and equally divided into 5-day group，10-day group，and 15-day group using a random number table. The left eye was set as a normal control eye，while the right eye was set as an experimental eye. The CNV model was established by subretinal injection of PEG. After model establishment，the eyeballs of mice were collected in each group. The sections of the retina were stained with hematoxylin-eosin（HE） to evaluate the CNV model. Thickness of the outer nuclear layer（ONL） in HE-stained sections of the retina was compared between the three groups to evaluate the retinal toxicity of PEG. The mRNA levels of CD146，vascular endothelial growth factor（VEGF），and vascular endothelial growth factor receptor 2（VEGFR2） in the neuroretina and RPE/choroid complex were determined by RT-PCR. Im－munohistochemical staining was used to measure the expression of CD146，VEGF，and VEGFR2 in the eyes of mice. Results：Both HE staining and thickness of the ONL confirmed that model establishment by subretinal injection of PEG was successful and reliable. The development of CNV was observed at 5 and 10 days after subretinal injection. The mRNA expression of CD146，VEGF，and VEGFR2 in the neuroretina and choroid was significantly higher in the experiment group than in the control group（F=30.412，P=0.000；F=84.974，P=0.000；F=117.423，P=0.000；F=918.786，P=0.000；F=319.110，P=0.000；F=113.896，P=0.000）. The Pearson correlation analysis showed that the expression of CD146 was positively correlated with the expression of VEGF and VEGFR2 in the RPE/choroid complex after subretinal injection of PEG（r=0.940，P=0.000；r=0.940，P=0.000；r=0.769，P=0.045；r=0.910，P=0.003；r=0.910，P=0.003；r=0.777，P=0.042）. The results of immunohistochemical staining showed that at 10 days after model establishment，the model group had significantly higher expression of CD146 and VEGFR2 in the ganglion cell layer，inner nuclear layer，outer plexi－form layer，and ONL than the normal control group（P=0.000，P=0.000，P=0.000）. Conclusion：The expression of CD146 is up-reg－ulated with the development of CNV and positively correlated with the expression of VEGF and VEGFR2. Therefore，CD146 may play a crucial role in the development of CNV.

Abstract:Objective：To investigate the differences in inflammation and autophagy in imiquimod（IMQ）-induced dermatitis model be－tween progranulin-deficient（PGRN-/-） mice and wild-type（WT） mice. Methods：The psoriasis-like dermatitis model was established by smearing the back skin of mice（44 WT mice and 16 PGRN-/- mice） with IMQ. Western blot，quantitative polymerase chain reac－tion(qPCR），hematoxylin and eosin（HE） staining were used to detect the changes in the tissue structure， inflammatory factors[（inter－leukin-6，IL-6），（interleukin-10，IL-10），（tumor necrosis factor-α，TNF-α），（nitric oxide synthase 2，NOS2），（cyclo-oxygen-ase 2，COX2），（interleukin-1β，IL-1β）]，apoptosis-related protein（cysteinyl aspartate specific proteinase 3，Caspase3） and autophagy-related proteins[（microtubule-associated protein 1 light chain 3，LC3），（ubiquitin binding protein，P62），（autophagy related protein 5，ATG5），（autophagy related protein 7，ATG7），（complex of autophagy related protein 5 and 12，ATG5-ATG12）] of the mice. Results：The mouse model of psoriasis-like dermatitis was successfully established. Significant thickening of epithelial tissues，increased infil－tration of inflammatory cells，and more angiogenesis were observed in the WT mouse model，with the highest expression levels of IL-6，IL-10，TNF-α，NOS2，and COX2 observed on the sixth day（P<0.05），and autophagy increased with time. In addition，the proportion of spleen increased significantly on the sixth day（P<0.05），and the expression levels of inflammatory factors IL-6，IL-10，TNF-α，NOS2，COX2，and IL-1β were highest（P<0.05）. Compared with the WT mouse model，the PGRN-/- mouse model showed more significant thickening of the back skin，higher expression levels of inflammatory factors IL-6 and NOS2（P<0.05），and signifi－cantly reduced levels of autophagy and apoptosis（P<0.05）. Conclusion：The dermatitis model is successfully established both in the WT mice and the PGRN-/- mice on the sixth day. The symptoms are more severe in the PGRN-/- mouse model than in the WT mouse model，which may be related to PGRN deficiency due to its role in impeding autophagy.

Abstract:Objective：To investigate the effects of hepatocyte growth factor（HGF）/c-Met-mediated endothelial progenitor cells（EPCs） on pulmonary artery pressure（PAP） and pulmonary vascular remodeling in rats with hypoxic pulmonary hypertension（HPH）. Methods：A rat model of HPH was established. Primary EPCs were iso－lated from the bone marrow of rats，cultured，and identified. The recombinant adenovirus plasmid carrying the c-Met gene was constructed and transfected into EPCs. The rats were divided into c-Met-EPCs group，EPCs group，pulmonary hypertension group，and normal group，with 10 rats in each group. c-Met-EPCs and EPCs were infused into the tail vein of rats in the c-Met-EPCs group and EPCs group，respectively，while normal saline was in－fused into the tail vein of rats in the pulmonary hypertension group and normal group. After normal feeding for a week，the mean PAP（mPAP） and right ventricular hypertrophy index（RVHI） were determined. The ultrastructural pathology of the pulmonary artery was observed under a transmission electron microscope，and the pathological sections of lung tissues were prepared to observe the histopathological changes of pulmonary arterioles. ELISA was used to determine the serum level of endothelin-1（ET-1），and the Griess method was used to determine the serum level of nitric oxide（NO）. Results：The measured values of mPAP for the c-Met-EPCs group，EPCs group，pulmonary hypertension group，and normal group were （23.17±3.07） mmHg，（30.85±3.15） mmHg，（33.55±5.47） mmHg，and （20.30±1.99） mmHg，respectively；the measured values of RVHI for the four groups were （32.48±2.38）%，（37.54±4.42）%，（38.53±2.81）%，and （26.05±3.05）%，respectively；the serum level of ET-1 for the four groups were （69.45±6.32） ng/L，（95.76±7.31） ng/L，（99.14±8.39） ng/L，and （62.35±6.06） ng/L，respectively；the serum level of NO for the four groups were （99.63±11.40） μmol/L，（56.07±3.32） μmol/L，（48.83±6.56） μmol/L，and （125.50±12.26） μmol/L，respectively. The mPAP，RVHI，and serum ET-1 level in the EPCs group and pulmonary hypertension group were significantly higher than those in the normal group（P<0.05），and the above-mentioned indices were significantly lower in the c-Met-EPCs group than in the EPCs group and pulmonary hypertension group（P<0.05）. The serum NO level in the EPCs group and pul－monary hypertension group was significantly lower than that in the normal group（P<0.05），and the serum NO level in the c-Met-EPCs group was significantly higher than that in the EPCs group and pulmonary hypertension group（P<0.05）. Transmission electron mi－croscopy showed obvious swelling of mitochondria and endoplasmic reticulum in pulmonary artery smooth muscle cells and dis－continuous distribution of endothelial cells in the EPCs group and pulmonary hypertension group，while swelling of mitochondria and endoplasmic reticulum in pulmonary artery smooth muscle cells was not obvious in the c-Met-EPCs group. The pathological sections of lung tissues showed obvious pulmonary vascular remodeling in the EPCs group and pulmonary hypertension group；however，the remodeling was reduced in the c-Met-EPCs group. Conclusion：HGF/c-Met-mediated EPCs can decrease PAP and improve pul－monary vascular remodeling in rats with HPH，possibly by inhibiting ET-1 release and promoting NO production.

Abstract:Objective：To investigate the temporal variation of corneal folds after death using the digital image analysis technique，and to screen out appropriate quantitative indicators. Methods：Rabbits were randomly divided into open-eye group and closed-eye group after hanging，with 20 rabbits in each group，and then they were placed in a dark room at a temperature of 20 ℃. A digital camera was used to capture rabbit corneal images at a one-hour interval within 72 hours after death. MATLAB software was used to obtain the im－ages of the pupil region of the cornea，and the four parameters of image texture features（CON，COR，ASM，and HOM） which reflected the postmortem change of corneal folds were extracted dynamically. The two groups were compared in terms of the variation trend of each parameter with postmortem interval（PMI），and the correlation between each parameter（y） and postmortem interval（x） was ana－lyzed. Results：CON increased with PMI and COR decreased with PMI in both groups，but compared with the closed-eye group，the open-eye group had significantly faster and greater changes. CON and COR were significantly correlated with PMI（P<0.05），and the regression equations were y=0.002 4x2-0.026 8x+0.467 3（CON，R2=0.978 in the open-eye group），y=0.000 4x2-0.010 6x+0.304 4（CON，R2=0.904 in the closed-eye group），y=-0.000 4x2+0.014 2x+3.893 8（COR，R2=0.938 in the open-eye group），and y=-3E-05x2+0.001 2x+3.980 8（COR，R2=0.852 in the closed-eye group）. ASM and HOM had no significant correlation with PMI（P>0.05）. Conclusion：The temporal variation of corneal folds is observed after death，and the texture features of CON and COR are well corre－lated with PMI.

Abstract:Objective：To investigate the effects of recombinant human elafin on the expression of inflammatory factors and nuclear factor-κB（NF-κB） p65 protein in neonatal mice with bronchopulmonary dysplasia（BPD） induced by chronic hyperoxia. Methods：A total of 30 C57BL/6J neonatal mice within postnatal 24 hours were equally and randomly divided into three groups：air group，hyper－oxia+Lactated-Ringer（L/R）-treated group（O2+L/R group，L/R solution as vehicle），and hyperoxia+elafin-treated group（O2+elafin group）. The O2+L/R group and the O2 +elafin group were exposed to 85% oxygen，while the air group was exposed to 21% oxygen（room air）. At 21 days of treatment，the baseline lung function was determined；HE staining was used to observe the morphological changes of the lung tissue；the mRNA expression of inflammatory factors was determined by real-time polymerase chain reaction and the expression of NF-κB p65 protein was determined by Western blot. Results：Compared with the air group，the O2+L/R group had a significantly lower body weight[（7.85±0.24） g vs. （5.33±0.63） g，P=0.000）] and delayed lung development；in addition，the O2+L/R group had significantly higher expression of tumor necrosis factor-α（TNF-α） mRNA（P=0.000），interleukin-1β（IL-1β） mRNA（P=0.000），and NF-κB p65 protein（P=0.001），but significantly lower expression of interleukin-4（IL-4） mRNA（P=0.000） and inter－leukin-13（IL-13） mRNA（P=0.000）. Compared with the O2+L/R group，the O2+elafin group had a significantly higher body weight（P=0.014） and relatively normal alveolar morphology；in addition，the O2+elafin group had significantly lower expression of TNF-α mRNA（P=0.011），IL-1β mRNA（P=0.000），and NF-κB p65 protein（P=0.001），but significantly higher expression of IL-4 mRNA（P=0.047） and IL-13 mRNA（P=0.000）. Conclusion：Elafin can effectively alleviate hyperoxia-induced lung injury in mice. The mechanism of action may be related to the inhibition of NF-κB activation and the release of inflammatory factors.

Abstract:Objective：To study the relativity between serine protease inhibitor（SerpinB3） and prostate cancer. Methods：Western blot was used to determine the SerpinB3 levels in 29 cases of prostate cancer tissues and 35 cases of prostate hyperplasia tissues，and im－munohistochemistry was used to detect the expression of SerpinB3 in 48 cases of prostate cancer tissues and 33 cases of prostate hy－perplasia tissues. The SerpinB3 small interfering RNA（siRNA） was transiently transfected into DU145 and PC3 cells by liposome method，including SerpinB3-siRNA1，SerpinB3-siRNA2 and SerpinB3-siRNA3. Random disturbance SerpinB3-siRNA-NC was used as negative control，and cells without transfection were blank control. SerpinB3-siRNA with the highest efficiency was chosen for sub－sequent cell migration assay in order to detect the effects of SerpinB3 on the invasion ability of DUl45 and PC3 cells. Results：Western blot results showed that the level of SerpinB3 protein in the tissues of prostate cancer was higher than that of the benign group（P<0.001）. Immunohistochemistry results showed SerpinB3 protein level was significantly higher in tumor tissues than that in benign tis－sues（P<0.001）. There was no statistic difference between SerpinB3 expression and the patient’s age and tumor metastasis（P=0.268，P=0.178，respectively），but SerpinB3 expression was significantly correlated with pathological grade and clinical stage of the tumor（P=0.031，P=0.042，respectively）. SerpinB3-siRNA could inhibit the proliferation and invasion of the cancer cells. Conclusion：Over expression of SerpinB3 can increase the invasion ability of prostate cancer cells.