含淤胆血清的培养体系体外诱导胚胎干细胞表达肝细胞功能的研究

Study of embryonic stem cells induced to express hepatic cell functions in vitro in a pathologic niche containing cholestatic serum

目的 探讨病理微环境体外诱导对小鼠胚胎干细胞(ESC)表达肝细胞功能的作用。 方法 悬滴培养ESC发育5-7 d的拟胚体,将其离散细胞种植于不同的分化体系,观察ESC在自主分化、肝细胞生长因子(HGF)或5％淤胆血清+HGF诱导下每天的分化情况(倒置相差显微镜),以及细胞的糖原、甘油三酯、白蛋白及尿素氮合成功能,细胞吲哚氰绿(ICG)和荧光二乙酯(FDA)染色的情况。 结果 ESC自主分化难以控制,分化为3个胚层的细胞。HGF促进ESC向内脏内胚层和中胚层(心肌)分化,但两者仅能表达低水平的肝细胞特异性功能。引入淤胆血清的HGF诱导体系中ESC能分化为较为均一的多角形细胞,具有较高水平的糖原、甘油三酯、白蛋白和尿素氮合成能力,ICG和FDA染色阳性。 结论 自主分化和HGF诱导ESC表达肝细胞代谢的能力有限。体外模拟病理性微环境可诱导ESC定向分化为肝系细胞,并具有较高水平的肝细胞代谢功能。

Objective To study the role of a pathologic niche inducing mouse embryonic stem cells (ESC) to express hepatic cell functions in vitro. Methods Embryoid bodies were developed from 5 to 7 day hanging-drop culture of mouse ESC, and their dissociated cells were planted in three differential systems: nothing added; with 20 ng/ml hepatooyte growth factor (HGF); and 5% rat cholestatic serum plus 20 ng/ml HGF added. Their differentiation was observed with inverted microscopes daily, and their hepatic functions were analyzed against their synthesis of glycogen, triglycerides, albumin, and urea nitrogen, and by their staining of indocyanine green (ICG) and fluorescein diacetate (FDA). Results ESC spontaneous differentiation was hardly being controlled to form three germ layers. HGF prompted the ESC to develop further into visceral endoderm and mesoderm (myocardium), but both of them only expressed a low level of hepatocyte-specific metabolic functions. With cholestatic serum added into the HGF-induced system, differentiated cells grew into similar angular cells, and had a higher level synthesis of glycogen, triglycerides, albumin and urea nitrogen with positive ICG and FDA staining. Conclusions Spontaneous or HGF-induced ESC differentiation has only limited hepatic functions expressed. A pathologic niche in vitro induces ESC to develop into hepatic lineages, with a higher level of hepatic metabolic functions.