一种改进的大鼠胎肝干细胞分离纯化方法

发布时间：2018-02-06 06:15

本文关键词： 肝干细胞细胞分离纯化免疫细胞化学鉴定　出处：《第三军医大学学报》2013年04期 　论文类型：期刊论文

【摘要】：目的改进大鼠胎肝干细胞的分离纯化方法,提高肝脏干细胞分离的纯度和活力。方法采用剪碎酶消化法分离大鼠胎肝干细胞,差速离心结合选择性消化法进行进一步纯化,含10%优等胎牛血清的DMEM/F12培养液培养。利用免疫细胞化学方法分别检测P1和P10代肝干细胞表面标志物。结果分离的大鼠胎肝干细胞接种培养瓶24 h后开始贴壁,5 d后细胞体积增大,呈梭形或多边形。经1～2次差异性消化传代后,细胞呈集落样生长,边缘清晰且折光率强。免疫细胞化学检测示:P1代大鼠胎肝干细胞C-Kit、甲胎蛋白(AFP)抗原、OV6、细胞角蛋白(CK)19呈阳性表达,不表达Alb。P10代肝干细胞C-Kit、CK19、OV-6及CD34呈阳性表达。结论大鼠胎肝干细胞分离纯化、培养扩增及鉴定方法简单易行。
[Abstract]:Objective to improve the method of isolation and purification of rat fetal liver stem cells and improve the purity and activity of liver stem cells isolation. Methods Rat fetal liver stem cells were isolated by shearing enzyme digestion. Differential centrifugation combined with selective digestion for further purification. DMEM/F12 medium containing 10% superior fetal bovine serum was cultured. The surface markers of P1 and P10 passage liver stem cells were detected by immunocytochemistry. Culture bottle 24. H then began to adhere to the wall. After 5 days, the cells increased in volume and appeared fusiform or polygon. After two different digestion and passage, the cells grew like colony. The results of immunocytochemistry showed that C-Kitt, AFP) antigen OV6 and cytokeratin CK-19 were positive. There was no positive expression of C-Kit19 CK19 OV-6 and CD34 in Alb.P10 generation liver stem cells. Conclusion the methods of isolation, amplification and identification of rat fetal liver stem cells are simple and feasible.
【作者单位】：南方医科大学附属解放军第458医院肝病防治中心;
【基金】：国家重点基础研究发展计划(973计划,2010CB945502)
【分类号】：R329-33

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