人胚胎主动脉血管内皮祖细胞的分离、培养及鉴定

Isolation,culture and identification of endothelial progenitor cells from human fetal aorta

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 isolate and culture endothelial progenitor cells （EPCs） from human fetal aorta, to analyze the capability of EPCs differentiating into endothelial cells, and evaluate the feasibility of using human fetal aorta-derived EPCs for clinical application of stem cell technology. Methods EPCs were isolated from clinical aborted fetal aorta by digesting aorta with type I collagenase, and proliferated in DMEM/F12 medium supplemented with LIF, bFGF, EGF, ECGS and low serum. The expression of CD133, CD34 and VEGFR2 of EPCs was assessed by RT-PCR, immunofluorescence staining and FACS analysis. EPCs were induced to differentiate into endothelial cells by cultured with medium containing 20% FCS and VEGF, and the differentiation ability was analyzed. Results EPCs derived from human fetal aorta expressed CD133, CD34 and VEGFR2. The EPCs proliferated rapidly in vitro in DMEM/F12 containing LIF, bFGF, EGF and ECGS, and possessed the ability of DiI-Ac-LDL uptake and tube formation on matrigel. After induction by VEGF in cultured EPCs, expression of CD133 was decreased significantly; and expression level of vWF, CD31 and ELAM-1 was increased. Moreover, EPCs＇ ability of tube formation and uptake of DiI-Ac-LDL was increased upon VEGF induction. All of these alteration indicated that EPCs had differentiated into endothelial cells. Conclusion EPCs derived from early fetal aorta possess strong self-renewal capability and the potential of differentiating into endothelial cells, indicating these cells could be used for clinical EPCs treatment.