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背景:目前各种诱导胚胎干细胞(ESC)分化为肝细胞的方法中,大多忽略了对分化细胞功能的诱导与鉴定。是否表达肝细胞功能应作为ESC向肝细胞分化的鉴定指标之一。目的:观察在肝细胞生长因子(HGF)体外诱导小鼠胚胎干细胞向肝细胞分化的体系中,瘀胆血清病理环境对分化细胞表达肝细胞功能的作用。设计:观察对比,体外细胞学实验。单位:中山大学附属第二医院肝胆外科。材料:实验于2004-10/2007-02在中山大学附属第二医院医学研究中心完成。小鼠E14ESC系由中山大学干细胞与组织工程中心提供;SD大鼠20只,鼠龄2周,购自中山大学动物实验中心。实验过程中对动物的处置符合动物伦理学要求。方法:对SD大鼠施以胆总管结扎切断手术,制作瘀胆模型,饲养10d后取全血制备瘀胆血清。用悬滴培养ESC发育5～7d的拟胚体,将其离散细胞种植于不同的分化体系,分别进行自主分化、20μg/L肝细胞生长因子、5%瘀胆血清+20μg/L肝细胞生长因子诱导分化。主要观察指标:①倒置显微镜下动态观察细胞形态变化。②分化4周时进行白蛋白、甲胎蛋白、CK18/19、糖原及吲哚氰绿和荧光二乙酯染色。③采用相应试剂盒每3天检测细胞合成白蛋白、三酰甘油及尿素氮功能。结果:①ESC自主分化难以控制,分化为3个胚层的细胞。肝细胞生长因子促进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。②引入瘀胆血清的肝细胞生长因子诱导体系中ESC能分化为较为形态均一的多角形细胞,其糖原、吲哚氰绿和荧光二乙酯染色均为阳性;白蛋白、三酰甘油和尿素氮合成能力显著高于自发分化和肝细胞生长因子诱导结果(P<0.05～0.01)。结论:采用瘀胆血清体外模拟病理性微环境可促进HGF诱导的ESC源性肝细胞表达高水平的肝特异性代谢功能。
BACKGROUND: At present, most of the methods for inducing embryonic stem cells (ESCs) to differentiate into hepatocytes are largely neglected to induce and identify differentiated cells. Whether the expression of hepatocyte function should be used as one of the identification indicators of differentiation of hepatocytes to ESC. OBJECTIVE: To observe the role of blood stasis and pathophysiology in hepatocyte differentiation induced by hepatocyte growth factor (HGF) in vitro to differentiate mouse embryonic stem cells into hepatocytes. Design: Observed contrast, in vitro cytology experiments. Unit: Second Affiliated Hospital of Sun Yat-sen University of hepatobiliary surgery. Materials: The experiment was performed at the Medical Research Center of the Second Affiliated Hospital of Sun Yat-sen University from October 2004 to February 2007. Mouse E14ESC was provided by Sun Yat-sen Stem Cell and Tissue Engineering Center. Twenty SD rats aged 2 weeks were purchased from Animal Experiment Center of Sun Yat-sen University. The handling of animals during the experiment conformed to the ethical requirements of animals. Methods: The SD rats were treated with common bile duct ligation to make the model of stasis. After 10 days of rearing, the whole blood was taken for the preparation of stasis bile serum. Embryoid bodies of embryonic day 5 ~ 7d were cultured in the hanging droplet, and their discrete cells were planted in different differentiation systems. The cells were divided randomly into 20μg / L hepatocyte growth factor, 5% stasis bile serum and 20μg / L hepatocyte growth Factor-induced differentiation. MAIN OUTCOME MEASURES: ① Inverted microscope dynamic changes in cell morphology. ② After 4 weeks of differentiation, albumin, alpha-fetoprotein, CK18 / 19, glycogen and indocyanine green and fluorescent diethyl ester were stained. ③ using the appropriate kit every 3 days detection of cell synthesis of albumin, triglyceride and urea nitrogen function. Results: ①It is difficult to control the differentiation of ESCs into three germ cells. Hepatocyte growth factor promotes the differentiation of ESC to visceral endoderm and mesoderm (myocardium), but both can only express low levels of hepatocyte-specific function. The introduction of stasis bile serum hepatocyte growth factor-induced system of ESC can differentiate into more uniform shape of polygonal cells, the glycogen, indocyanine green and fluorescent diethyl ester staining were positive; albumin, triglyceride and The urea nitrogen synthesis ability was significantly higher than that induced by spontaneous differentiation and hepatocyte growth factor (P <0.05 ~ 0.01). CONCLUSION: The stasis-free cholangitis serum can simulate the pathological microenvironment in vitro and promote HGF-induced high level of liver-specific metabolic function in ESC-derived hepatocytes.