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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。
Objective: To investigate the feasibility of inducing embryonic stem cells (ESCs) to differentiate into hepatocytes in the “pathological microenvironment” culture system of cholestasis. Methods: Mouse ESC cell line E14 was cultured in leukemia inhibitory factor medium to differentiate into embryoid body spontaneously. FGF - 4 and HGF were added initially to induce embryonic body, then placed in the pathological microenvironment The culture medium was further cultured for 2 weeks. Then the morphology of the cells was observed, the albumin and CK8 / 18 immunohistochemical staining, the albumin and thyroid-transferred protein RT-PCR, the glycogen staining and the urea synthesis were analyzed. Results: The primary differentiated ESCs were cultured in the “pathological microenvironment” screening medium with 5% cholestatic serum. The initial cell growth was inhibited. After 2 weeks, the cells differentiated into hepatocyte - like cells. The cells showed good homogeneity. Immunohistochemical staining showed that albumin and CK8 / 18 expression; RT-PCR showed albumin, thyroid transglutaminase mRNA transcription; cells have glycogen and urea synthesis. CONCLUSION: The pathological microenvironment culture system containing cholestatic serum was used to effectively screen out the competent hepatocytes from embryonic stem cells initially induced by FGF - 4 and HGF. The cells showed good homogeneity and were suitable candidates for clinical hepatocyte replacement Treatment, access to a rich source of donor cells provides a new idea.