山羊内皮祖细胞的分离与诱导分化

Isolation and induced differentiation of endothelial progenitor cells from goat

目的研究山羊外周血来源的内皮祖细胞的分离与诱导分化及鉴定方法。方法采用密度梯度离心法分离山羊外周血单核细胞，然后将细胞接种在纤维连接蛋白包被的培养瓶中。EGM-2MV培养基培养，分别在第4、7、10天进行细胞鉴定。第14天做透视电镜观察。结果培养24h后，可见部分网形大单核细胞黏附于培养瓶底，第4天细胞由圆形转变为梭形贴壁细胞，呈条索状或管状分布。免疫组化及透视电镜证实细胞为正在分化的内皮祖细胞。结论山羊的外周血可培养出大量的内皮祖细胞，能满足动物实验研究的需要，且数量级达1×10^6-1×10^7。

Objective To investigate the methods of isolation, cultivation and identification of endothelial progenitor cells （EPCs） from goat peripheral blood. Methods The mononuclear cells were isolated from goat peripheral blood by density gradient centrifugation, and then the ceils were plated on fibronectin-coated culture flask. The cells were suspended in EGM-2MV medium. Identification by immunocytochemistry was performed at day 4, 7 and 10 after cultivation. Then the cells were scanned by transmission electron microscopy （TEM） to confirm EPCs lineage at day 14 after cultivation. Results Some round large mononuclear cell adhered to the culture flask at 24 hours after cultivation, fusiform attached cell from round cells distributed as cord or duct shape at day 4. These cells were confirmed as the EPCs which were differentiating by immunocytochemistry and TEM. Conclusion Lots of EPCs could be cultured in vitro from goat peripheral blood. The cell number of 1 × 10^6 - 1× 10^7 coud meet the needs of animal experiment.