人外周血内皮祖细胞分离培养与鉴定

Isolation, cultivation and identification of endothelial progenitor cells from human peripheral blood

目的探讨人外周血内皮祖细胞体外分离培养和诱导分化的实验方法,为其在神经系统疾病中的应用提供理论依据。方法抽取健康成年男性志愿者外周血10ml,密度梯度离心获得单个核细胞,经体外培养后于倒置相差荧光显微镜下观察细胞生长情况及形态变化;免疫荧光染色检测细胞表面标志物CD133、CD34和凝血因子Ⅷ表达水平;流式细胞术检测CD133-FITC和CD34-PE双阳性细胞所占比例及CD34-PE单阳性细胞所占比例。结果倒置相差荧光显微镜下观察,人外周血单个核细胞经体外培养至第2天时,呈贴壁生长;至第4～6天时细胞集落形成,集落周围有内皮样细胞,细胞质和细胞膜呈红色和绿色双色荧光,细胞核呈蓝色荧光,提示内皮祖细胞特异性吸附Dil标记的乙酰化低密度脂蛋白和FITC标记的Ⅰ型荆豆凝集素,具有内皮细胞功能。免疫荧光染色CD133、CD34和凝血因子Ⅷ表达阳性;流式细胞术检测CD133-FITC和CD34-PE双阳性细胞所占比例为38.30%,CD34-PE单阳性细胞所占比例为82.60%。结论初步确立人外周血内皮祖细胞体外分离培养和诱导分化的实验方法,为内皮祖细胞的移植研究奠定了基础。

Objective To explore the procedures of isolation, cultivation and identification of endothelial progenitor cells （EPCs） from human peripheral blood, and to provide theoretical foundation for the application in nervous system diseases. Methods Ten milliliters of peripheral blood were collected from healthy male adult volunteers. The mononuclear cells were isolated from human peripheral blood by density gradient centrifugation and were cultured in vitro. The growth and morphological changes were observed by inverted phase-contrast fluorescence microscope. The expressions of EPCs specific markers such as CD133, CD34 and coagulation factor Ⅷ were assessed by immunofluorescent staining. The ratio of fluorescein isothiocyanate labeled CD133 （CD133-FITC） and phycoerythrin labeled CD34 （CD34-PE） double-positive cells and the ratio of CD34-PE single-positive cells were determined by flow cytometry. Results Under the observation of inverted phase-contrast fluorescence microscope, the cells attached to the wall after 2 d cultivation, exhibited the colony after 4-6 d cultivation. There were endothelium-like cells around the colony. The cytoplasm and cell membrane presented red and green bicolor fluorescence, and the cell nuclear presented blue fluorescence. The cells specifically absorbed Dil labeled acetyl-low density lipoprotein （Dil-Ac-LDL） and FITC labeled ulex europaeus agglutinin Ⅰ （FITC-UEA-Ⅰ）, and presented epithelial cells function. Immunofluorescent staining showed that the cultivated cells were positive for CD34, CD133 and coagulation factor Ⅷ. The flow cytometry indicated that the ratio of CD133-FITC and CD34-PE double-positive cells was 38.30%, and the ratio of CD34-PE single-positive cells was 82.60%. Conclusion The primary establishment of isolation, cultivation and identification of EPCs from human peripheral blood, indicates that EPCs from human peripheral blood can be isolated, cultivation and identification in vitro. It will be the basis for the research on EPCs transplantation.