人外周血内皮前体细胞的分离与体外培养

Isolation of Endothelial Progenitor Cells from Human Peripheral Blood and Induction of Differentiation by Ex Vivo Expansion

目的研究体外诱导培养外周血来源内皮前体细胞(endothelial progenitor cells,EPCs)的可行性并检测其表型和功能。方法用密度梯度离心法从人外周血中分离单个核细胞。在加有胎牛血清(fetal blood serum,FBS)、血管内皮生长因子(vascular end othelial growthfactor,VEGF)和碱性成纤维生长因子(basic fibroblast growthfactor,bFGF)的M199培养液中培养。用流式细胞仪检测其表面标志CD34和KDR的表达情况。通过荆豆凝集素-1(ulex europaeus agglutinin-1,UEA-1)结合试验和乙酰化低密度脂蛋白(acetylatedlowdensitylipoprotein,acLDL)吞噬试验确定其内皮细胞功能。结果培养过程中可观察到典型的内皮细胞。培养7天后,粘附细胞表达CD34和KDR,阳性率分别为29.5%±9.9%和33.8%±9.2%。粘附细胞的UEA-1结合试验和acLDL吞噬试验均为阳性。结论从成人外周血中可分离到EPCs,并能在体外增殖分化为内皮细胞。体外培养是获得EPCs移植和体外组织工程技术所需要的足够EPCs数量的一条途径。

Objective To investigate the feasibility of inducing endothelial progenitor cells(EPCs) derived from human peripheral blood cells into endothelial cells in vitro and test their phenotype and function.Methods Mononuclear cells were isolated from human peripheral blood cells by density gradient centrifugation and cultured in M199 medium with fetal blood serum(FBS),vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF).The CD34 and KDR of endothelial cells were detected by flow cytometry.The function of endothelial cells was determined by the experiment of ulex europaeus agglutinin-1(UEA-1) adsorption and acetylated low density lipoprotein(acLDL) internalization.Results Typical morphology of endothelial cells(ECs) was observed.After 7 days of culture,the attached cells expressed CD34 and KDR,and the positive ratios were 29.5%±9.9% and 33.8%±9.2% respectively.The results of FITC labeled UEA-1 adsorption and Dil-acLDL internalization of the attached cells were both positive.Conclusion EPCs can be derived from the peripheral blood cells of adult individuals,expanded in vitro,and committed into an endothelial lineage in culture.This culture methodology may be suitable to produce the high endothelial cell counts desired in EPCs transplantation and ex vivo tissue engineering approaches.