中文摘要：

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。

英文摘要：

BACKGROUND ： Recently, little attention has been paid to how to induce and identify the functions of differentiated cells in the methods for embryonic stem （ES） cells differentiation into hepatocytes. Whether the differentiated cells express functional characteristics of hepatocytes should be one of the markers to identify the hepatic differentiation of ES cells, OBJECTIVE： To direct mouse embryonic stem cells in vitro differentiation into functional hepatocytes by introduction of murine cholestatic serum in hepatocyte growth factor （HGF）-induced system. DESIGN ： A controlled observation and in vitro cytological trial SETTING： Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Sun Yat-sen University MATERIALS： The experiment was carried out in the Medical Research Center of the Second Affiliated Hospital of Sun Yat-sen University from October 2004 to February 2007. The mouse E14 ES cell line was kindly provided by the Stem Cell and Tissue Engineering Center of Sun Yat-sen University. Twenty male SD rats, aged 2 weeks, were purchased from the Experimental Animal Center of Sun Yat-sen University. All animal experimental procedures were abided by the rules of animal ethnics. METHODS： The SD rats were undergone common bile duct ligation to induce cholestasis. Ten days after the operation, the whole blood of rats was collected to prepare cholestatic serum. The ES cells were cultured using hanging-drop method for 5-7 days to develop embryonic bodies （EBs）. The dissociated EBs cells were then induced hepatic differentiation with spontaneous system, HGF （20 μg/L） system and cholestatic serum （5%） plus HGF （20 μg/L） system, respectively. MAIN OUTCOME MEASURES： The cellular morphologic changes were observed using transverse microscopy dynamically. （2） The cell staining for albumin, α-fetopretein, CK18/19, glycogen, indocyanine green （ICG） and fiuorescein diacetate （FDA） was done after 4 weeks differentiation, （3） The hepatocyte-specific metabolic