| Objective:To establish and evaluate a mouse model of cholestatic cirrhosis and to investigate the changes of TNF-?,TGF-?1 and IL-6 in this model,and their influences on the differentiation of hepatic stem cells.Methods:1.Balb/c mice were subjected to Bile Duct Ligation(BDL),serum biochemical parameters,hepatic HE staining and Masson staining were used to evaluate the modeling.2.Immunohistochemistry,western blot,Real-Time PCR were used to detect the changes of TNF-?,TGF-?1,IL-6 in the modeling.3.Different concentrations of TNF-?,TGF-?1,IL-6 were applied to infect HP14-19,whose expressions of ck18 and ck19 were evaluated by western blot,Real-Time PCR,immunofluorescence.PAS staining was used to evaluated the glycogen synthesis capacity.Cck-8 proliferation assay was used to evaluated the cytotoxicity of cytokines.Results:1.In the BDL groups,blood biochemical parameters were significantly increased(P<0.05),HE staining showed hepatocytes necrosis,Masson staining showed increased collagen fibers.2.Real-Time PCR and western blot showed that the expressions of TNF-?,TGF-?1 and IL-6 were significantly higher in the experimental groups than that in the control group(P<0.05).Immunohistochemistry showed that TNF-?,TGF-?1,IL-6 were diffusely expressed in hepatic lobules,TNF-? and IL-6 were expressed in the portal area,and TGF-?1was mainly expressed in the sinus space.3.There was no significant differentiation difference in the expressions of ck18 and ck19 between control group and that induced by TNF-?,TGF-?1,IL-6 for 3 days.After 5-days,inductions,the expressions of ck18 and ck19 in TNF-?,TGF-?1,IL-6 most concentration groups were higher than that of the control group(P<0.05).The expressions of ck18 in TNF-?,IL-6 20,40,80 ng/mL concentration groups were higher than that of ck19(P<0.05),and the TNF-? had a higher expression of ck18 than IL-6(P<0.05).While the TGF-?1 groups were the opposite,the expressions of ck19 in 20,40,80 ng/mL concentration groups were higher than that of the ck18(P<0.05).The immunofluorescence suggested that TNF-? had a higher expression of ck18,and TGF-?1 had a higher expression of ck19.PAS staining revealed that TGF-?1 had a strong glycogen synthesis function.After 10 days,inductions,the expression trends of ck18 and ck19 were the same as that induced for 5 days,and western blot,PAS staining immunofluorescence were stroner,while Real-Time PCR were weaker in the expressions of ck18 and ck19.The results of cck-8 suggested that TNF-? and TGF-?1 had no influence on the proliferation of HP14-19.Conclusion:Common bile duct ligation can successfully imitate cholestatic cirrhosis.TNF-?,TGF-?1,IL-6 are elevated in this disease,the functions of TNF-? and IL-6 in the differentiation of hepatic stem cells are similar,they mainly promote the differentiation of liver stem cells into hepatocytes,while TGF-?1 mainly promotes the differentiation of that into colangiocytes.TNF-? and TGF-?1 had no significant effect on the proliferation of hepatic stem cells. |
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