aFGF、HGF体外诱导小鼠ESC向肝细胞定向分化及标志物研究

Study of hepatic differentiation in vitro from mouse ESC induced with a FGF and HGF and the expression of hepatic biomakers

目的观察酸性成纤维细胞生长因子(acid fibroblast growth factor,a FGF)、肝细胞生长因子(hepatocyte growth factor,HGF)对小鼠胚胎干细胞(embryonic stem cell,ESC)体外定向诱导分化作用及诱导后肝细胞标志物的表达水平。方法体外培养小鼠ESC使其发育成拟胚体,然后加入a FGF、HGF诱导ESC定向分化成肝细胞。收集培养上清液,RIA法测定甲胎蛋白(alpha fetoprotein,AFP)、白蛋白(albumin,ALB)浓度。PCR和免疫印迹法检测ALB、细胞角蛋白8(CK8)以及细胞角蛋白18(CK18)在细胞内mRNA和蛋白表达水平。结果 ESC培养5 d后发育成为拟胚体,加入不同浓度a FGF继续培养,5 d后AFP浓度降低,ALB浓度升高,与对照组相比有显著性差异。细胞内ALB、CK8及CK18表达水平明显升高。一次诱导后加入HGF,继续诱导5 d,上清液中AFP浓度降低,ALB浓度升高,具有浓度依赖性。ALB、CK8及CK18在细胞内表达升高。结论体外培养小鼠ESC,加入a FGF、HGF后可诱导其向肝细胞定向分化。肝细胞标志物AFP水平明显降低,ALB、CK8以及CK18表达水平明显升高。

Objective To observe the effects of aFGF and HGF on the differentiation of embryonic stem cells （ESC）,and the expression of hepatic markers after induction.Methods Embryoid was cultured and developed into ESC bodies,then were induced with aFGF and HGF.The concentration of AFP and ALB in the supernatants were detected by radio immuno-assay.The mRNA and protein expression of ALB,CK8 and CK18 were determinated by PCR and Western blotting.Results After adding different concentrations of aFGF,the concentration of AFP decreased while the ALB concentration increased in these induced groups with the concentration dependence of aFGF,and showed significantly difference compared with the control group.The expression of ALB,CK8 and CK18 in cells increased.After induction with aFGF for 5 days,HGF was added for the second induction.Five days later,the concentration of AFP and ALB in the supernatant were continue decreased or increased with concentration dependence.The expression of ALB,CK8 and CK18 increased in induced groups.Conclusion In vitro distribution of aFGF and HGF in the culture of mouse embryonic stem cells can induce the directional differentiation to liver cells.Liver cell markers AFP decreased,while ALB,CK8 and CK18 obviously increased.