TGF-?1-induces Activation Of HSC-T6 And Epithelial-mesenchymal Transition In Rats

Objective:To observe the effect of TGF-?1 on activation and epithelial-mesenchymal transition,to investigate the effects of TGF-?1 on TGF-?1/Smads signaling pathway in rat hepatic stellate cells-T6,reveal the pathogenesis of hepatic fibrosis,and provide new target for the treatment of hepatic fibrosis and liver circosis.Methods:Adopt the MTT method to screening the optimum working concentration of TGF-?1 in vitro HSC-T6 cultured.After the hepatic stellate cells were stimulated by TGF-?1 of 10 ?g/L for 24 hours,the morphology of the cells was observed under inverted phase contrast microscope,the expression of F-actin which on behalf of cotoskeletal structure was detected by Immunofluorescence staining,the expression of a-SMA and N-cadherin mRNA,vimentin mRNA,E-cadherin mRNA as well as Smad4 mRNA werre measured by RT-qPCR;The protein changes of a-SMA and EMT relative marker protein of N-cadherin,vimentin,E-cadherin and Smad4 were assessed by Western blot after different concentrations(0?g/L,5?g/L,10 ?g/L)of TGF-?1 interventing HSC-T6 for 24 hours.Results:In our study,the MTT data showed that it had optimal cell survival rate when l0?g/L TGF-(31 dealt with cells for 24 hours.After HSC-T6 were treated with TGF-?1,cells stretched,pseudopodia increased and turn into stellate,cells connections were looser,so that represented a significantly activated state under inverted phase contrast microscope;Immunofluorescence results revealed there were F-actin filaments gathered to form stress and distributed along the long axis of the cells;RealTime-qPCR results showed that the expression of a-SMA mRNA,vimentin mRNA and Smad4 mRNA in experimental group were significantly higher than the control group(P<0.05),while E-cadherin mRNA was obviously lower than the control group(P<0.05),the expression of Smad4 mRNA was a little bit higher whereas no significantly than the control group.Western blot demonstrated that the protein expression of a-SMA,N-cadherin,vimentin and Smad4 were in dose-dependent increased while E-cadherin was in dose-dependent decreased comparing with the control group under the effect of different concentration of TGF-?1(P<0.05).Conclusion:1.TGF-?1 might induce activation and epithelial-mesenchymal transition of HSC-T6 of rats,and its effect was concentration dependent.2.Up regulation of Smad4 expression in HSC-T6 cells after stimulation with TGF-?1 TGF-?1/Smads signaling pathway may be involved in cell activation and epithelial mesenchymal transition of HSC-T6 cells.