CD133免疫磁珠分选脐血内皮祖细胞的培养及鉴定

Culture and identification of endothelial progenitor cells from cord blood with CD133 immunomagnetic sorting

目的:建立具备高效增殖、血管生成与迁移能力的脐血内皮祖细胞(endothelial progenitor cell,EPC)分离培养鉴定方法。方法:应用免疫磁珠分选纯化脐血单个核细胞中的CD133+细胞,体外EGM-2MV培养液培养扩增,通过形态学、细胞表面标志及细胞功能鉴定EPCs,并与脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)作比较。结果:EPCs分离培养7d左右开始出现小集落,21d左右集落扩大、相互融合,并呈现出典型铺路石样改变;培养14d左右,约90%的细胞免疫荧光Dil-ac-LDL和FITC-UEA-1双阳性,阳性率达90%。CD133和CD34阳性率从86.04%降至2.96%、90.88%降至2.99%,而CD31阳性率从1.12%升至99.88%。在增殖、血管生成与迁移能力上比较,EPCs明显优于HUVECs(P<0.05)。结论:通过CD133免疫磁珠分选脐血单个核细胞,可培养出具有高效增殖、血管生成与迁移能力的EPCs。

Objective：To establish a method for isolating and culturing endothelial progenitor cells（EPCs）, which have high potential of proliferation, migration and angiogenesis, from cord blood. Methods： CD133^＋ cells were selected from fresh cord blood mononuclear cells by magnetic activated cell sorting system （MACS）, and were cultured in EGM-2MV medium. EPCs were identified by examining the morphology, cell markers and functions. And the EPCs were compared with human umbilical vein endothelial cells （HUVECs）. Results： On the 7^th day, the adherent cells exhibited the small colonies; and on the 21^th day,the colonies were expanded,confluenced and displayed a typical ＂cobblestone＂ morphology. On the 14^th day, 90% attached cells took up Dil-ac-LDL, and bound FITC-UEA-1 （double positive fluorescence）. The cell markers of CD133 and CD34 decreased from 86.04% to 2.96% and 90.88% to 2.99%, respectively, while CD31 increased from 1.12% to 99.88%. Compared with HUVECS, EPCs had more potent potential of proliferation, migration and angiogenesis（P〈0.05）. Conclusion： CD133^＋ MACS can be used to isolate EPCs, with high capacity of proliferation, angiogenesis and migration, from cord blood mononuclear cells.