利用自体血清培养人外周血内皮祖细胞

Culture of human peripheral blood endothelial progenitor cells by autologous serum

背景:传统方法培养人外周血来源内皮祖细胞操作复杂,费用大,细胞获得率较低。目的:利用自体血清培养人外周血来源内皮祖细胞,并鉴定其功能。方法:采用密度梯度离心法从人外周血分离得到单个核细胞,体外培养分化为内皮祖细胞。按培养基条件不同分为EGM-2MV组、添加自体血清组(M199+体积分数10%自体血清+胰岛素样生长因子)、添加胎牛血清组(M199+体积分数10%胎牛血清+血管内皮细胞生长因子+碱性成纤维细胞生长因子+胰岛素样生长因子+表皮生长因子)。观察内皮祖细胞增殖、迁移能力;采用细胞形态观察、双荧光染色法及流式细胞仪等技术对培养的内皮祖细胞进行鉴定。结果与结论:培养第7天,EBM-2MV组和添加自体血清组细胞增殖能力和迁移率都优于添加胎牛血清组(P<0.05)。每组细胞经结合Dil标记的乙酰化低密度脂蛋白和异硫氰酸荧光素标记的荆豆凝集素双色荧光染色鉴定后双阳性率>80%,免疫细胞化学检测显示每组细胞CD133,CD34和KDR的表达均为阳性。证实在M199培养液中添加自体血清是一种简单、高效的培养内皮祖细胞方法。

BACKGROUND： Traditional method for culture of endothelial progenitor cells from human peripheral blood is complex to operate, costs high, and acquires a small number of cells. OBJECTIVE： To culture endothelial progenitor cells from human peripheral blood using autologous serum and identify their functions. METHODS： Peripheral blood was collected from volunteers. Mononuclear cells were separated by density centrifugation and were induced to differentiate into endothelial progenitor cells in vitro. The influences of different culture conditions （EGM-2MV, M199＋10% autologous serum＋insulin like growth factor, M199＋10% fetal bovine serum＋vascular endothelia growth factor＋basic fibroblast growth factor＋insulin-like growth factor＋epidermal growth factor） on the proliferation and migration of endothelial progenitor cells were observed. The cultured endothelial progenitor cells were identified by cell morphology observation, fluorescent staining and flow cytometry. RESULTS AND CONCLUSION： Endothelial progenitor ceils with higher proliferation and migration ability were obtained under EGM-2MV and M199＋10% autologous serum ＋ insulin-like growth factor conditions （P 〈 0.05）. Furthermore, the percentage of double positive cells stained by Dil-labeled acetylated low density lipoprotein and FITC-labeled Ulex europaeus agglutinin 1 was higher than 80%, and these cells also expressed CD34, CD133 and KDR. Addition of autologous serum to M199 culture medium is a simple, highly efficient method for culturing endothelial progenitor cells.