摘要目的 探讨体外定向诱导小鼠胚胎干细胞(ES细胞)分化为肝细胞的方法及肝损伤模型肝内移植的可行性.方法 常规培养ES细胞后,继续悬浮培养4 d以形成拟胚体(EBs),转移EBs到铺有明胶的6孔板中贴壁培养,并添加3 mmol/L丁酸钠开始诱导分化,7 d后加入淤胆血清筛选、纯化ES源性肝细胞,分化过程中用光学显微镜和电子显微镜观察细胞形态及超微结构的改变;用逆转录-聚合酶链反应(RT-PCR)方法检测肝细胞特异性标志基因:白蛋白(ALB)、甲胎蛋白(AFP)、甲状腺素运载蛋白(TTR)、α1抗胰蛋白酶(AAT)、葡萄糖6磷酸酶(G6P)、酪氨酸转氨酶(TAT)mRNA水平的表达;以荧光示踪剂CFDA-SE标记诱导获得的肝细胞,并移植到肝损伤小鼠肝内,观察移植细胞在肝内的定居、增殖情况.结果 诱导分化过程中,ES细胞形态逐渐出现肝细胞样改变,其超微结构与小鼠肝细胞超微结构十分相似;RT-PCR结果显示,随着诱导时间的推进,标志肝细胞发育过程的ALB、AFP、TTR、AAT、G6P、TAT mRNA顺序表达;肝内移植实验结果显示:ES源性肝细胞可在肝损伤小鼠肝内定居并增殖.结论 丁酸钠联合淤胆血清可以诱导ES细胞分化为肝细胞,ES源性肝细胞肝损伤模型体内移植是可行的,这有可能为细胞移植治疗难治性肝病提供一种新的细胞来源.

abstractsObjective To explore the feasibility of embryonic stem (ES) cells-derived hepatocyte transplantation into hver-injured mouse liver. Methods Mouse ES cells were induced to differentiate into hepatocytes using sodium butyrate and cholestatic serum. During the course of cell differentiation, cell mor-phological changes and hepatic-specific genes were detected. The resulting ES cells-derived hepatocytes were labelled with CFDA-SE followed by transplantation into liver-injured mouse liver. Then the recipient liver was removed,sliced,and immunofluorence staining of ALB was performed. The hver slice was examined u-sing confocal laser scanning microscopy. Results During the course of ES cell differentiation induced by so-chum butyate and cholestatic senun, numerous epithelial cells resembling hepatocytes were observed. Elec-tron micrographs revealed that the differentiated cells were rich in endoplasmic reticulum, ribosomes, mito-chondria,and glycogen,which were typical uhrastructural features of mature hepatocytes. RT-PCR analysis showed that these hepatocyte-like cells expressed hepatic-specific genes. Immunofluorence staining of ALB (red fluorence) showed that CFDA-SE labelled ES cells-derived hepatocytes (green fluorence) could settle down,proliferate in recipient liver,and no teratoma formation was found two weeks after transplantation.Conclusion In vivo transplantation of ES cells-derived hepatocytes into liver-injured mouse liver is feasible and safe,which may provide a new cell source for cell transplantation treatment in liver dieases.