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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 gene on normal differentiation of Fetal liver stem cells (FLSCs) and its related mechanism. Methods FLSCs were enriched by three-step separation method. The mRNA and protein expressions of AFP, ALB, Tg737 and HNF4α were detected by RT-PCR and Tg737 and HNF4α mRNA at different time points. Transfection of FLSCs was used to observe the inhibitory effect of Tg737 after transfection. After inhibition of Tg737, RTPCR was used to detect the mRNA expression of AFP and ALB at different induction time points. Western blot was used to detect the protein expression of HNF4α after inhibition of Tg737. Results FLSCs were efficiently enriched by three-step separation method. The mRNA expression of AFP gradually decreased while the expression of ALB mRNA and protein of Tg737 and HNF4α gradually increased in HGF-induced FLSCs . The mRNA and protein expression of Tg737 were down-regulated by shRNA. There was no significant change in the mRNA level of AFP and ALB in Tg737-treated group during HGF induction. The protein expression of HNF4α was significantly down-regulated after Tg737 inhibition. Conclusion Tg737 and HNF4α are involved in the normal differentiation of FLSCs. The lack of expression of Tg737 can inhibit the normal differentiation of FLSCs. The mechanism of its inhibition may be through the down-regulation of HNF4α.