大鼠肝卵圆细胞的分离培养及干细胞标志分析.DOCVIP

一种改进的大鼠胎肝干细胞分离纯化方法 王亚萍 佟明华 雷香丽 孔祥平 罗显荣（510600广州，南方医科大学：附属解放军第四五八医院肝病防治中心) ［提要］　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 简单易行的大鼠胎肝干细胞分离纯化、培养扩增及鉴定方法，为深入研究肝干细胞生物学特性和进行体内移植实验提供了物质基础。 【关键词】　　肝干细胞　　细胞分离　纯化 免疫细胞化学 鉴定 An Improved Method for the Isolation and Purification of Rat Fetal Liver Stem Cells Wang Yaping, Tong Minghua, Lei Xiangli, Kong Xiangping，Luo Xianrong （458 Hospital of PLA Affiliated to Southern Medical University, Southern Medical University，Guangzhou, 510600）Abstract］　Objective To improve the isolation and purification methods of rat fetal liver stem cells(LSCs), and therefore to further improve the purity and vitality of the isolated LSCs. Methods　Use shear-enzyme-digestion method to isolate rat fetal liver stem cells, and then adopt differential centrifugation combined with selective-digestion method for further purification, including 10% master fetal calf serum DMEM/F12 nutrient solution culture, using Immunohistochemistry method to detect the surface markers of P1 and P10 LSCs respectively. Results　 The isolated rat LSCs in inoculation culture bottle initially attached after 24 hours, the cells increased in size after 5 days, with a spindle shape or polygon. By 1 ~ 2 times difference digestion transfer of culture, the cells grow in colony sample, with clear margin and strong refractive index. With the Immunohistochemistry method ,we can draw this conclusion: P1 generation of rat fetal LSCs C-Kit, alpha-fetoprotein (AFP) antigen, OV6, cytokeratin (CK)19 take on positive attributes, while ALB doesnt .p10 generation of LSCs CD34, C-Kit,OV6,CK19 take on positive attributes.Conclusions　The methods of isolating, purifying, cultivating, amplifying and appraising rat fetal LSCs a