| ObjectiveTo investigate the expression of oval cells in the liver of children with biliary atresia,establishment and evaluation of cholestatic cirrhosis model induced by bile duct ligation in mouse,Transplantation of hepatic stem cells in spleen to investigate the differentiation of HP14-19 in cholestatic cirrhosis and to investigate the effect of SDF-1/CXCR4 and SDF-1/CXCR7 on proliferation,migration and anti-oxidative stress of hepatic stem cells.Methods1.100 children with biliary atresia in our hospital were randomly selected to detect the serum alpha fetal protein(alpha-fetoprotein,AFP)level,20 patients with choledochus cyst were selected as the control,the levels of AFP were compared between the two groups..The expression of the oval cells marker ov-6,c-kit in the liver of the patients with biliary atresia was detected by immunohistochemistry,the liver tissues of patients with choledochus cyst were selected as the control.2.84 balb/c mice were randomly divided into the experimental group(n=56)and the control group(n=28).The experimental group was induced by bile duct ligation through midline incision,and the control group underwent sham operation.The general condition and liver structure was observed after operation,serum TBIL,ALT,DBIL,AST were detected by automatic biochemistry analyzer in 1,3,5,7,14,,21,28 day separately,liver tissue morphology were observed by H&E staining,expression of collagen fiber and a-SMA,CK-19 protein in live tissue of 28 d was detected by masson staining and immunohistochemistry3.Spleen transplantation HP14-19 cells which labeled by RFP-luc to cholestatic cirrhosis mice model,the control group of infused PBS,In vivo imaging was used to detect the colonization of the cells in the liver to determine the feasibility of the spleen transplantation.HP14-19 cells labeled by PKH26,The engraftment and differentiation were detected by fluorescence microscopy and confocal microscopy.4.Recombinant adenovirus which containing CXCR4 or CXCR7 gene was applied to infect HP14-19,respectively.Flow cytometry and Western blot were used to detect the expression of CXCR4 or CXCR7 as membrane receptors.Cell proliferation activity was detected by MTT assay.Transwell assay was carried out to evaluate the SDF-1 induced migration ability of HP14-19 with CXCR4 or CXCR7 overexpression.HP14-19 was exposed to H2O2 to establish oxidative injury model.The cell viability with CXCR4 or CXCR7 overexpression was measured by MTT assay.Microplate Reader was to evaluate the LDH and SOD levels.Results1.Serum AFP was significantly increased in 92 children with biliary atresia,and the control group was in normal range;the expression of ov-6,c-kit in liver tissue samples with biliary atresia was positive compared to that in control group.2.Compared with the control group,the general condition in the experimental group were generally poor,The gallbladder and common bile duct expansion,cholestasis.Serum biochemical parameters were significantly higher(P<0.05),H&E staining indicated that the structural of the liver and bile duct proliferation.Masson staining and immunohistochemical staining demonstrated that the positive expression of collagen fiber,a-SMA and CK-19 were significantly higher than that in control group after 28 d of duct ligation than that in control.3.In vivo imaging and fluorescence microscopy showed that HP14-19 which transplant from spleen was express in damage liver,confocal microscopy has not been observed the HP14-19 express biliary epithelial cell marker CK-19.4.The expression of CXCR4 and CXCR7 receptoron HP14-19 was significantly higher after infection with adenovirus.Overexpression CXCR7 but not CXCR4 can enhance the proliferation of HP14-19.The cellwith CXCR4 or CXCR7 overexpression significantly enhanced SDF-1 induced cell migration and cell survival rate of oxidative stress,which CXCR7 showed strong effect on the migration.Compared with the control group,overexpression of CXCR4 or CXCR7 could reduce the LDH level and increased SOD activity caused by H2O2.ConclusionsOval cell is exist in liver of children with biliary atresia,and play an important role in the procession of cholestatic cirrhosis.Mouse cholestatic cirrhosis model can be established successfully by bile duct ligation through midline incision,liver stem cells can be successfully transplanted into the injured liver via spleed,but not differentiated into bile duct epithelial cells.Overexpression of CXCR4 was involved in the proliferation of HP14-19 mediated by SDF-1,both CXCR4 and CXCR7 mediated the SDF-1 induced migration and anti-oxidative stress of HP14-19. |
PDF Full Text Request