【摘要】 目的 :探讨淤胆血清“病理微环境”培养体系诱导胚胎干细胞 (ESC)向肝细胞分化的可行性。方法 :将小鼠ESC细胞系E14在无白血病抑制因子培养基中培养 ,使其自发分化为拟胚体 ,加入FGF - 4和HGF初步诱导 ,然后置于 5 %淤胆鼠血清“病理微环境”筛选培养液中继续培养 2周 ,然后进行细胞形态学观察 ,白蛋白和CK8/ 18免疫组化染色 ,白蛋白与转甲状腺蛋白RT -PCR检测 ,细胞糖原染色及尿素合成功能分析。结果 :经初步诱导分化的ESC置于 5 %淤胆血清“病理微环境”筛选培养液中培养 ,初期细胞生长受抑制 ,2周后分化为肝细胞样细胞 ,细胞呈现良好的均质性 ;免疫组化染色显示白蛋白和CK8/ 18表达 ;RT -PCR显示有白蛋白、转甲状腺蛋白等的mRNA转录 ;细胞有糖原和尿素合成功能。结论 :采用含淤胆血清的病理微环境培养体系从经FGF - 4和HGF初步诱导的胚胎干细胞中有效筛选出了具有功能的肝细胞 ,细胞有较好的均质性 ,为临床肝细胞替代治疗、获取丰富供体细胞来源提供了新思路。更多还原

【Abstract】 AIM: To investigate whether a pathological micro-environmental culture system consisting of cholestatic sera induces embryonic stem cells (ESC) to differentiate into hepatocyte-like cells in vitro, and select hepatic stem cells from differentiating embryonic stem cells. METHODS: Mouse ESC, E14 cell line, were cultured in Dulbecco’s modified Eagle’s medium containing 106 U/L recombinant mouse leukemia inhibitory factor (rmLIF) and 10% FCS. After embryonic bodies formed by the hanging drop culture method, they were exposed to fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF) for one week, and then placed to a pathological micro-environmental culture system consisting of 5% cholestatic sera and cultured for 2 weeks. Morphological examination, immunocytochemical staining of albumin and CK8/18 were carried out, and mRNA level of albumin and transthyretin were detected by RT-PCR. Glycogen storage and urea synthesis of the cells were tested with PAS staining and colorimetric assay, respectively. RESULTS: The proliferation of cells was inhibited at the early stage when cultured in a pathological micro-environmental culture system consisting of 5% cholestatic sera, but 2 weeks later, a large number of epithelial-like cell colonies were observed, which exhibited hepatocellular phenotype, expressing albumin and CK8/18, transcribing mRNA of albumin and transthyretin and synthesizing glycogen and urea. CONCLUSION: A pathological micro- environmental culture system consisting of 5% cholestatic sera could not only induce embryonic stem cells to differentiate into hepatocyte-like cells, but select hepatic stem cells from differentiating embryonic stem cells initially induced by FGF-4 and HGF in vitro as well.更多还原