人外周血来源的血管内皮祖细胞体外诱导分化

目的研究外周血来源内皮祖细胞的分离、培养、分化及鉴定方法。方法采集正常人外周血单个核细胞进行体外培养，通过免疫组化、免疫荧光和流式细胞术测定分化细胞表面特异性抗原的表达及其变化。结果培养7d后多数细胞贴壁生长，逐渐显示内皮细胞的形态特点，传代培养4周后呈典型铺路石样，并可继续稳定传代扩增；免疫组化和免疫荧光染色CD31、CD34、血管内皮细胞生长因子受体．2和vWF均呈阳性；流式细胞显示，经体外诱导培养后CD34^＋／AC133^＋细胞数明显增多。结论外周血来源的单个核细胞中含有能分化成血管内皮细胞的内皮祖细胞，其在一定条件下可稳定分化、扩增。

In vitro induction and differentiation of human endothelial progenitor cells from peripheral blood and their characteristics

Objective To study the methods of isolation, culture, differentiation and identification of endothelial progenitor cells(EPC) from peripheral blood. Methods Total hPBMNCs were isolated from peripheral blood of human volunteers by Histopaque-1077 density-gradient centrifu-gation. The cells were suspended in endothelial basal medium supplemented with EGM-2-MV-SingleQuots. Cultured cells were subjected to immunohistochemistry and immunofluorescence to analyze the expression of CD31, CD34, VEGFR-2 and vWF. FACS was performed to count the number of CD34+/AC133+ cells. Results EPCs exhibited clonal morphology and cobblestone morphology after 7 days or 4 weeks culture respectively. They were positive stain for CD31, CD34, vascular endothelia growth factor receptor 2 and vWF. The number of CD34+/AC133+ cells increased significantly after 2 weeks culture. Conclusion EPCs can be obtained from hPBMNCs and expanded enough for harvesting in vitro under certain condition.