人脐血内皮祖细胞的体外分离和培养

目的建立一种稳定的体外分离和培养人脐血来源内皮祖细胞的方法。方法采用密度梯度离心法从人脐带血中分离单个核细胞,将其接种至人纤维连接蛋白包被的六孔板中,用EGM-2培养基诱导培养。通过形态学观察、细胞表面特异性抗原、摄取功能和体外血管形成能力对内皮祖细胞进行鉴定。结果细胞形态学观察发现,刚分离的单个核细胞较小,呈圆形,4 d后可见少量的圆形和梭形贴壁细胞,8 d后有明显集落形成,14 d后相邻集落相互融合,呈现出典型铺路石样改变。内皮祖细胞能摄取乙酰化低密度脂蛋白,结合荆豆凝集素1,表达CD34、CD133和血管内皮细胞生长因子受体2,并且具有体外血管生成能力。结论采用密度梯度离心法可从人脐带血中成功分离和培养出内皮祖细胞,以用于相关实验研究。

In vitro isolation and cultivation of endothelial progenitor cells from human umbilical cord blood

Objective To establish a stable method for isolation and cultivation of endothelial progenitor cells（ EPCs） from human umbilical cord blood. Methods Mononuclear cells（ MNCs） were isolated from human umbilical cord blood by density gradient centrifugation,then they were seeded in 6-well plates pre-coated with fibronectin（ Fn） and cultured in EGM-2. EPCs were identified by morphology,cell surface markers,ability to uptake Ac-LDL and bind UEA-1 and in vitro tube formation characteristic.Results The morphology of freshly isolated MNCs was small and spherically shaped. A small amount of adherent cells with circular,oval and spindle shapes appeared after 4 days culture. Colonies formed after 8days culture. The cells exhibited typical cobblestone morphology after 14 days culture. Some cells formed network structures and linear cord-like structures. EPCs could uptake Ac-LDL,bind UEA-1,express cell surface markers,CD34,CD133 and VEGFR-2,and possess in vitro tube formation ability. Conclusions EPCs can be successfully isolated by density gradient centrifugation from human umbilical cord blood and cultivated for further research.