四种表面标志物分子在大鼠骨髓源性内皮祖细胞分离鉴定中的应用

The application of four surface markers in isolation and identification of rat bone marrowderived endothelial progenitor cells

【目的】研究血小板-内皮细胞黏附分子（platelet endothelial cell adhesion molecule-1,PECAM-1/CD31）、CD34、CD133和血管内皮生长因子受体2（vascular endothelial growth factor receptor 2,VEGFR-2）四种表面标志物分子在大鼠骨髓源性内皮祖细（endothelial progenitor cells,EPCs）的分离鉴定中的应用。【方法】采用Ficoll密度梯度离心法分离提取骨髓单个核细胞,体外诱导培养。观察细胞形态的动态变化,流式细胞技术检测CD31、CD34、CD133和VEGFR-2四种细胞表面标志物的表达、荧光双染Dil-ac-LDL与FITC-UEA-I等方法对EPCs进行鉴定。【结果】分离培养的EPCs在3~4 d时,光学显微镜下可见部分贴壁细胞集落样生长,呈圆形或不规则形;7~10 d,贴壁的梭形细胞从细胞团边缘出芽生长;14~21d可观察到梭形细胞首尾相连成条索状结构,梭形细胞基本融合,融合后逐渐呈铺路石状。细胞培养至7 d时,Dil-ac-LDL/FITC-UEA-I双阳性细胞率大于85%,流式细胞术结果显示：EPCs四种表面分子CD31、CD34、CD133和VEGFR-2表达阳性。【结论】密度梯度离心法结合检测CD31、CD34、CD133和VEGFR-2四种表面标志物的表达等技术,可用于分离鉴定稳定增殖分化的内皮祖细胞,其生物学特征明显。

【Objective】To investigate four surface markers molecules of CD31, CD34, CD133 and VEGFR-2 in the application of the isolation and identification of the rat bone marrow derived endothelial progenitor cells（EPCs）.【Method】Bone marrow mononuclear cells were isolated using Ficoll density gradient centrifugation methods and cultured in vitro. Then EPCs were identified by dynamic change of cell morphology, Dil-Ac-LDL uptake and FITC-UEA-1 binding assay, expression of four surface markers using flowcytometry.【Result】The bone marrow-derived EPCs cultured for 3~4 days appeared to form foci or cord-like structures; in 7~10 d, adherent spindle cells formed by being budded off the edge; in 14~21 d, spindle cells mainly fused together to cobble-stone morphology. After 7 days of culture, adherents EPCs were characterized by immunofluorescence and FACS. The majority of cells（over 85%） stained positive for DilAc LDL and lectin, and expressed endothelial/stem cell markers, including CD31、CD34、CD133and VEGFR-2, confirming the cell type of EPCs.【Conclusion】Density gradient centrifugation combined with detection of the four surface markers（CD31, CD34, CD133 and VEGFR-2） could be used to separate and identify EPCs and these cells had obvious biological characteristics.