Tg737基因参与胎肝干细胞正常分化的机制研究

The effect and mechanism of Tg737 on differentiation of fetal liver stem cells

目的探讨Tg737基因对胎肝干细胞(Fetal liver stem cells,FLSCs)正常分化的影响及相关机制。方法三步分离法富集FLSCs;在HGF诱导FLSCs的不同时间点,RT-PCR检测AFP、ALB、Tg737及HNF4α的mRNA表达,Western blot检测Tg737和HNF4α的mRNA和蛋白表达;慢病毒Tg737-shRNA转染FLSCs,观察转染后Tg737的抑制效果;Tg737抑制后,RTPCR检测不同诱导时间AFP和ALB的mRNA表达;Western blot检测Tg737抑制后HNF4α的蛋白表达。结果三步分离法能有效富集FLSCs;在HGF诱导FLSCs的过程中,AFP的mRNA表达呈逐渐降低的趋势,ALB的mRNA表达逐渐升高,Tg737及HNF4α的mRNA和蛋白表达均呈逐渐升高的趋势;shRNA能有效下调Tg737的mRNA和蛋白表达;在HGF诱导过程中,Tg737抑制组AFP和ALB的mRNA水平均无明显变化;Tg737抑制后HNF4α的蛋白表达显著下调。结论 Tg737和HNF4α参与了FLSCs的正常分化,Tg737的表达缺失会抑制FLSCs的正常分化,其抑分化机制可能是通过下调HNF4α来实现的。

Objective To investigate the effect of Tg737 on differentiation of fetal liver stem cells （FLSCs）and to explore the molecular mechanism involved in the procedure.Methods FLSCs were obtained from embryonic-day （ED）-14 rat fetuses by a procedure previously reported by our group.At different points in being stimulated by HGF,mRNA expression of AFP,ALB,Tg737 and HNF4α were detected by RT-PCR,protein level of Tg737 and HNF4α were tested by Western blot at the same time.Commercial short hairpin RNA （shRNA） lentiviral particles was transfected into FLSCs,RT-PCR and Western blot were used to detect gene scilencing effect on Tg737.After inhibiting Tg737,mRNA level of AFP and ALB were detected by RT-PCR at different time points via HGF stimtdation.Protein expression of HNF4α was tested by Western blot as well.Results FLSPCs were isolated successfully from ED-14 rat liver and cultured as described in our previous report.During FLSCs were stimulated by HGF,mRNA expression of AFP presented a gradually reduced trend,meanwhile,the expression of ALB,Tg737 and HNF4α at mRNA level increased step by step.In addition,protein level of Tg737 and HNF4α were elevated also.shRNA-Tg737 could down-regulate both mRNA and protein level of Tg737 in FLSCs efficiently.During FLSCs were stimulated by HGF,mRNA expression of AFP and ALB had no significant change in Tg737 inhibited group.Protein level of HNF4α presented remarkable down-regulation along with Tg737 inhibition.Conclusion Tg737 and HNF4α participate in the normal differentiation of FLSCs.Tg737 RNA interference may lead to the dedifferentiation of FLSCs via down-regulate HNF4α.