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目的研究人胚胎血管内皮祖细胞(EPCs)分离、扩增及鉴定的方法,并评价其分化成血管内皮细胞的能力,为人胚胎血管来源EPCs作为干细胞技术治疗疾病的细胞材料提供依据。方法从14周龄流产人胚胎主动脉中应用胶原酶消化法分离获得EPCs,用含有碱性成纤维细胞生长因子、表皮生长因子和白血病抑制因子的低血清培养基体外扩增培养EPCs。分离培养的EPCs鉴定采用细胞免疫荧光染色、RT-PCR及流式细胞术,检测EPCs细胞的特异标志CD133、CD34和血管内皮细胞生长因子受体2(VEGFR2)。培养的EPCs应用VEGF进行诱导分化,并评价其分化成为血管内皮细胞的能力。结果分离的人胚胎主动脉EPCs细胞表达EPCs的标志分子CD133、CD34和VEGFR2。EPCs在体外特定低血清培养条件下表现很强的增殖能力。培养的EPCs细胞经过VEGF诱导后,细胞表达CD133明显降低,表达vWF、CD31和ELAM-1增强,并且体外成管能力和摄取Ac-LDL能力增强,表明细胞分化成为血管内皮细胞。结论人胚胎早期主动脉的EPCs具有很好的体外自我更新能力和分化成为血管内皮细胞的潜能,可作为EPCs治疗疾病的细胞材料。
OBJECTIVE: To study the method of isolation, amplification and identification of human embryonic endothelial progenitor cells (EPCs) and to evaluate their ability to differentiate into vascular endothelial cells, and to provide basis for human embryonic vascular derived EPCs as cell material for stem cell therapy. Methods EPCs were isolated from the aorta of 14-week-old human embryos by collagenase digestion. EPCs were cultured in vitro with low serum medium containing basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor. EPCs were isolated and identified by immunofluorescence staining, RT-PCR and flow cytometry. The specific markers CD133, CD34 and VEGFR2 were detected by flow cytometry. The cultured EPCs were induced to differentiate using VEGF and evaluated for their ability to differentiate into vascular endothelial cells. Results The isolated human embryonic aorta EPCs cells expressed EPCs markers CD133, CD34 and VEGFR2. EPCs show strong proliferative capacity under specific low serum culture conditions in vitro. The expression of CD133, vWF, CD31 and ELAM-1 were enhanced in cultured EPCs cells induced by VEGF, and the ability of in vitro tube formation and uptake of Ac-LDL was enhanced, indicating that the cells differentiated into vascular endothelial cells. Conclusion EPCs of human early embryo aorta have good self-renewal ability in vitro and their potential of differentiation into vascular endothelial cells, which can be used as the cell material for the treatment of diseases by EPCs.