小鼠胚胎肝干细胞体外向肝样细胞的诱导分化

背景：目前肝干细胞尚无特异性标志物，故其分离、培养尤其是纯化技术尚不成熟。目的：观察体外培养的小鼠胚胎肝干细胞生物学特性，及其向肝样细胞的诱导分化能力。设计、时间及地点：细胞学体外观察，于2008-01/06在厦门大学附属中山医院消化中心实验室完成。材料：SPF级BALB/c胎鼠20只，13.5 d龄，由厦门大学生命科学学院实验动物中心提供。方法：无菌操作取出胎鼠肝脏，用细胞刮梳理后用IV型胶原酶消化。取分离纯化后的第2代细胞进行免疫荧光染色。细胞培养到第3代时，分别用3 mmol/L丁酸钠盐与0.1% DMSO进行诱导，5 d后收集细胞进行Western blotting实验。主要观察指标：胚胎肝干细胞的形态及表面分子的表达，诱导后胚胎肝干细胞mRNA与蛋白水平的变化。结果：分离培养的细胞第2代即可得以纯化，呈克隆样生长，高表达白蛋白、甲胎蛋白、C-met及角蛋白19，且多数细胞共表达白蛋白与角蛋白19、C-met与角蛋白19，双阳性率约80%。细胞诱导后肝细胞标志物白蛋白表达明显增加，而作为不成熟肝细胞的经典标志物甲胎蛋白表达减少，同时干细胞标志物 c-kit mRNA水平也明显下降，角蛋白19水平无明显变化。结论：胚胎肝干细胞具有双向分化潜能，经丁酸钠盐与DMSO诱导后可以向肝样细胞分化。

Differentiation of mouse fetal hepatic stem cells into hepatocyte-like cells in vitro

BACKGROUND: There are no specific markers of hepatic stem cells (HSCs), so its isolation, culture and purification technique is immature. OBJECTIVE: To study the biological characterization of mouse fetal HSCs and differentiation of HSCs into hepatocytes in vitro. DESIGN, TIME AND SETTING: The cytology in vitro study was performed at the Digestion Center Laboratory of Zhongshan Hospital Affiliated to Xiamen University from January to June 2008.MATERIALS: A total of 20 SPF level 13.5-day BALB/c pregnant mice were supplied by Animal Experimental Center, School of Life Sciences, Xiamen University. METHODS: Fetal mouse liver was sterilely obtained, combed using cell scrape, and digested in type IV collagenase. At passage 2, cells were identified by immunofluorescence staining following isolation and purification. At passage 3, cells were induced by 3 mmol/L sodium butyrate and 0.1% dimethyl sulfoxide for 5 days. Cells were harvested for Western blotting assay. MAIN OUTCOME MEASURES: Morphology and surface markers expression of HSCs were measured. The mRNA and protein level changes of HSCs after induction were identified.RESULTS: Cell was purified and grew into colonies by cultivation in vitro. Cells highly expressed albumin, alpha-fetoprotein, C-met and keratin-19. Most cells co-expressed albumin and keratin-19 or C-met and keratin-19. Double positive rate was about 80%. Albumin increasingly expressed following induction, but alpha-fetoprotein decreasingly expressed. Simultaneously, c-kit mRNA levels were significantly reduced. No significant changes were found in keratin-19 levels. CONCLUSION: HSCs show the capacity for bipotent differentiation, and can differentiate into hepatocyte-like cells following sodium butyrate and dimethyl sulfoxide treatment.