体外诱导人脐血间充质干细胞向肝细胞样细胞分化的研究

Inducing differentiation of human umbilical cord blood mesenchymal stem cells into hepatocyte-like cells in vitro

目的 探讨人脐血间充质干细胞能否在体外诱导分化为肝细胞样细胞,并探索其定向诱导分化为肝细胞样细胞的分化机制。方法 无菌条件下采集正常产妇脐血,用相对密度为1.077的淋巴细胞分离液分离脐血MNC,进而采用贴壁培养法获得MSC,流式细胞仪检测其表面际志。分别用HGF、FGF4、俩者联合、无生长因子四种处理因素,及含2％FBS的DMEM,1×ITS讲行诱导培养,并于诱导前及诱导后的第7、14、21、28天留取细胞,RT～PCR法检测AFP、白蛋白及C-met FGFR2mRNA的表达,免疫细胞化学法检测AFP、白蛋白、抗肝细胞抗体和CK18的表达,PAS法进行糖原染色,分析是否诱导出肝细胞样细胞。结果 MSC强表达CD29、CD44,不表达CD34。诱导后7,21天分别检测出AFP,白蛋白mRNA及蛋白的表达,28天检测出CK18、抗肝细胞抗体的表达,21天、28天均可检测到糖原染色阳性细胞,随诱导时间的延长C-met,FGFR2 mRNA表达增高,HGF+FGF4诱导组肝系细胞标志阳性率均高于单独生长因子诱导组(P<0.05);单独生长因子诱导组间无明显差异(P>0.05)。无生长因子诱导组在以上时间点均未检测到上述指标。结论HGF、FGF4均能诱导人脐血MSC分化为具有肝系细胞表型和功能的细胞,且二者在一定程度上有联合作用。生长因子与其相幢受体之间,可能存在正反馈调节机制。

Objective To investigate whether human umbilical cord blood mesenchymal stem cells would be able to differentiate into hepatocyte - like cells in vitro, and explore the mechanism of inducing differentiation.Methods Human umbilical cord blood samples were obtained sterilely from healthy parturients. MNCs were isolated by lymphocyte separation medi-um(density 1 .077g/ml) , MSCs were obtained by adhere culture. The surface antigen expression of MSCs were detected by flow cytometry. MSCs were induced with DMEM medium,containing 2%FBS, 1×ITS,and HGF alone, FGF4 alone, HGF + FGF4 or no growth factor respectively. The markers of hepatic linear cells, C - met and FGFR2 were examined before induction , and after 7, 14, 21 and 28 day induction by methods of RT - PCR, immunocytochemistry and PAS in every group . Results MSCs expressed CD29 and CD44 strongly, but did not express CD34. AFPmRNA and protein were detected on day 7 after induction. Albumin mRNA and protein, CK18 ,hepatocyte antigen, glycogen were detected on day 21 after induction . The expression of C-met and FGFR2 increased with induce culture.The markers of hepatocyte linear did not appear without growth factor. The positive percent of every hepatocytic marker at the same time point had statistically difference between HGF + FGF4 and HGF alone or FGF4 alone( P < 0 .05) . Conclusion Both HGF and FGF4 can induce umbilical cord blood MSC to differentiate into hepatocyte - like cells singly or coordinately. There may be positive regulation mechanism between growth factor and its corresponding receptors.