小鼠胎肝干细胞的分离培养与鉴定

背景：胎肝细胞可能具有比骨髓干细胞等更强的增殖分化能力和更低的免疫原性，但目前涉及胎肝干细胞直接分离、培养的报道甚少。 目的：拟在体外分离培养小鼠胎肝干细胞，并对其生物学特性进行初步鉴定。 设计、时间及地点：细胞学体外观察，于2008-03/06在重庆市神经病学重点实验室完成。 材料：SPF级13.5 d龄昆明种胎鼠9只，由重庆医科大学实验动物中心提供。 方法：采用胶原酶+EDTA联合消化法与差速贴壁法体外分离胎鼠肝干细胞，按2×108 L-1接种，待细胞80%~90%汇合后消化传代。采用链霉亲和素-生物素-过氧化酶复合物技术对原代接种后5 d的贴壁细胞进行多种肝干细胞表面标志物的标记。 主要观察指标：原代胎肝干细胞形态变化，胎肝干细胞的传代扩增情况，胎肝干细胞表面标志的表达。 结果：原代培养24 h细胞贴壁，呈致密圆形，边缘清楚；3 d左右部分细胞呈梭形，7 d后细胞铺展呈上皮样；传代后细胞扩增速度无明显变化，至第5代仍保持较均一的上皮细胞状。原代接种后5 d的贴壁细胞，人干细胞因子受体与甲胎蛋白呈阳性表达，白蛋白与细胞角蛋白19呈阴性。 结论：胎肝干细胞原代培养早期表达甲胎蛋白与人干细胞因子受体，不表达白蛋白和细胞角蛋白19，提示所分离的胎肝干细胞可能是一种较原始的干细胞，尚处在未分化的早期阶段。

Isolation, incubation and identification of mouse embryonic hepatic stem cells

BACKGROUND: Fetal liver cells can have stronger abilities to proliferation and differentiation and lower immunogenicity compared to bone marrow stem cells. However, there are few studies on direct isolation and culture of embryonic hepatic stem cells (EHSCs). OBJECTIVE: To isolate and cultivate EHSCs in vitro and to identify their biological features. DESIGN, TIME AND SETTING: The cytology in vitro controlled study was performed at the Chongqing Key Laboratory of Neurology from March to June 2008. MATERIALS: A total of 9 SPF Kunming fetal mice aged 13.5 days were obtained from Animal Experimental Center of Chongqing Medical University. METHODS: Collagenase + EDTA digestion and differential adherence method were used to isolate EHSCs, which were then incubated at 2×108 /L. Cells were digested and passaged when 80%-90% cells were confluent. Using streptavidin-biotin-peroxidase complex technique, adhered cells following 5 days of incubation were labeled with various EHSC surface marker. MAIN OUTCOME MEASURES: Morphology, passage, amplification and surface marker surface of EHSCs were measured. RESULTS: The isolated EHSCs adhered to the culture plastic and presented pykno-round cells and distinct borderline 24 hours after cultivation in vitro. Cells grew spindle-shaped 3 days. After 7 days they grew like epithelium. Cell amplified speed following passage did not have significant changes. Cells still presented epithelium-like shape at the passage 5. The adhered cells at day 5 following primary incubation were positively for human stem cell factor receptor and alpha fetoprotein, and negatively for albumin and cytokeratin 19. CONCLUSION: EHSCs were positively for human stem cell factor receptor and alpha fetoprotein, and negatively for albumin and cytokeratin 19 in early primary culture. This indicated that the cultivated cells are proved to be primordial progenitor cells and still in undifferentiated early phase.