人脐带间充质干细胞体外诱导分化为肝细胞样细胞及ERK1/2通路活化研究

目的探讨人脐带间充质干细胞(hucMSC)体外诱导分化为肝细胞样细胞(HLC)的机制。方法用组织块贴壁培养法分离、纯化和扩增hucMSC,观察细胞形态,流式细胞术检测细胞表面标志;经肝细胞生长因子(HGF)/碱性成纤维细胞生长因子(bFGF)/抑瘤素M(OSM)联合诱导后,用RT-PCR、免疫荧光法检测肝细胞和hucMSC中特异基因AFP、ALB、TDO、CK18和ABCG2、CD44、OCT4及ALP、AFP、CK18蛋白的表达;用western blot检测经诱导后的hucMSC中ERK1/2信号通路活化情况。结果流式细胞术结果表明,分离培养的hucMSC表达干细胞特异性标志CD13、CD29、CD44,不表达造血干细胞标志CD34、CD45和HLA-DR;RT-PCR结果表明,诱导后的hucMSC ALB、AFP、TDO、CK18基因表达增强,ABCG2、CD44、OCT4表达降低;免疫荧光结果表明,hucMSC可在体外诱导分化为具有肝系细胞表型细胞,其ALP、AFP、CK18蛋白表达增强;western blot结果表明诱导后hucMSC的ERK1/2磷酸化水平增加。结论 hucMSC可在体外诱导分化为HLC,其机制可能与ERK1/2磷酸化相关。

Transdifferentiation of human umbilical cord-derived mesenchymal stem cells into hepatocyte-like cells in vitro and activation of ERK1/2 pathway

Objective To explore the mechanism of differentiation of human umbilical cord-derived mesenchymal stem cells(hucMSCs) into hepatocyte-like cells(HLCs) in vitro.Methods hucMSCs were isolated,purified and amplified from fresh human umbilical cord with tissue adherent culture method.The morphology of hucMSCs was observed and the cell surface markers were determined by flow cytometry.After hucMSCs were induced with hepatocyte growth factor(HGF),basic fibroblast growth factor(bFGF) and oncostatin M(OSM),the expressions of hepatocyte specific genes,e.g.,AFP,ALB,TDO and CK18,and hucMSC specific genes,including ABCG2,CD44 and OCT4,were detected by RT-PCR.The expressions of AFP,ALB and CK18 proteins were detected by immunofluorescence method.The activation of ERK1/2 pathway was investigated with Western blotting.Results The isolated hucMSCs expressed human MSC-specific markers CD13,CD29 and CD44,but no hematopoietic stem cell markers CD34,CD45 and HLA-DR.After hucMSCs were induced,the expressions of AFP,ALB,TDO and CK18 genes in hucMSCs increased,but ABCG2,CD44 and OCT4 genes decreased.Immunofluorescence demonstrated hucMSCs were differentiated into HLCs with increased ALB,AFP and CK18 proteins in vitro,and the increased phosphorylation of ERK1/2 was detected in the induced hucMSCs by western blotting.Conclusion hucMSCs may be induced into HLCs in vitro by a mechanism associated with phosphorylation of ERK1/2.