体外诱导小鼠骨髓间充质干细胞分化为肝样细胞

目的:体外诱导小鼠骨髓间充质干细胞分化(BMSCs)为肝样细胞,提高分化效率.方法:将第一代BMSCs随机分为诱导组和对照组.诱导组加肝细胞生长因子、成纤维生长因子、表皮生长因子、抑瘤素M等进行诱导培养,观察细胞形态,检测甲胎蛋白(AFP)、白蛋白(A1b)、细胞角蛋白18(CK18)、酪氨酸氨基转移酶(TAT)和细胞色素P450 2b9(CYP2b9)的mRNA表达,A1b合成以及A1b和CK18蛋白标记细胞阳性率.结果:诱导组第3天出现多边形细胞,5—7d上皮样细胞呈岛状分布,14 d呈铺路石状.对照组细胞为长梭形.第7,14,21天,诱导组细胞AFP,A1b,CK18 mRNA和A1b蛋白检测阳性;第14,21天,细胞表达TAT和CYP2b9 mRNA.对照组除AFP mRNA呈弱阳性外,其余均为阴性.第7,14,21天,诱导组CK18阳性率分别为71.4%.75.9%,80.6%:A1b阳性率分别为75.0%,79.7%.81.1%.而对照组第7天CK18和A1b阳性率仅2.3%,1.7%,与诱导组相比有显著差异(P_(CK18)=1.97×10~(-5),P_(A11b)=3.08×10~(-6)).结论:BMSCs在体外可以被诱导分化为肝样细胞,诱导率最高可达80%以上.

Differentiation of mouse bone marrow mesenchymal stem cells into hepatocyte-like cells in vitro

AIM:To induce hepatocyte-like cells from mouse bone marrow mesenchymal stem cells,and improve their differentiation rate in vitro. METHODS:Bone marrow mesenchymal stem cells were collected from C57 mice,and cultured in differentiation medium including HGF,FGF4, EGF,OSM,Dex and ITS.Expression of hepato- cyte specific genes,such as AFP,Alb,CK18,TAT and CYP2b9 was detected by RT-PCR.Cells expressing Alb and CK18 proteins were identi- fied by immunofluorescence staining and flow cytometry. RESULTS:In the differentiation group,some polygonal ceils appeared on day 3 in culture, cobblestone-shaped epithelial-like cells ap- peared after 5 to 7 days and increased in number until day 14,while control group cells retained their spindle morphology.AFP,Alb and CK18 mRNA could be detected on days 7,14 and 21. TAT and CYP2b9 mRNA were also detected on days 14 and 21.However,Alb,CK18,TAT and CYP2b9 mRNAs were not detected in the con- trol group,and only low levels of AFP mRNA were detected.In the differentiation group,Alb protein was detected by immunofluorescence on days 7,14 and 21.The percentages of cells that were Alb-positive were 75.0%,79.7% and 81.1% on days 7,14,21 respectively,and the percent- ages that were CK18-positive were 71.4%,75.9% and 80.6%,respectively. CONCLUSION:Bone marrow mesenchymal stem cells can differentiate into hepatocyte-like cells in vitro,which provides a solid experimen- tal basis for hepatocyte transplantation.