摘要
目的:探讨人外周血内皮前体细胞(endothelialprogenitorcellsEPCs)体外诱导分化为内皮细胞(ECs)的生长增殖规律及其特性,以期证实人外周血是临床治疗缺血性疾病一个理想的内皮前体细胞来源。方法密度梯度离心提取单个核细胞,接种在人纤连蛋白包被的培养板上,培养于含有促血管生长因子VEGF、b-FGF、IGF、EGF等的内皮生长基质EGM-2MV中。每天倒置显微镜观察,并记录。4d后去除未附着细胞,继续培养黏附细胞,同时,黏附细胞用Dil-AC-LDL和FITC-UEA-1进行免疫荧光染色,荧光显微镜和共聚焦激光扫描显微镜观察。收集第7d的黏附细胞,流式细胞仪检测细胞表面标志CD34和CD31。结果接种1d后,一些细胞变形;2d后,有细胞团形成;3d后,细胞团周围一些贴壁细胞开始出现;4d后,黏附细胞呈短梭形或多角形贴壁生长,荧光显微镜、共聚焦激光扫描显微镜下可观察到Dil-AC-LDL和FITC-UEA-1双阳性的黏附细胞;第6d、7d,黏附细胞呈长梭形;FACS分析,CD34和CD31阳性率分别为(14.13±2.79)%、(54.67±3.44)%。结论外周血中确实存在EPCs,并且在促血管生长因子VEGF、b-FGF、IGF、EGF等的刺激下能分化为成熟的内皮细胞。
Background and Objective： The present aim is to study the isolation, inducement and identification of endothelial progenitor sells （EPCs） from peripheral blood mononuclear sells （PBMCs）, and to probe into its biological activity in vitro for the angiogenesis therapy. Methods： Total mononuclear sells （MNC） were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin coated culture dishes, and cultured in EGM-2 MV, Which contains VEGF, bFGF, IGF, EGF. After incubation for 4days, the nonadherent cells were removed, and fresh culture medium was applied. At the same time, adherent sells which were double positive for Dil-AC-LDL-up take and lectin binding by direct fluorescent staining were observed under fluorescent and confocal laser microscope respectively .On the seventh days, Adherent sells were further identified by demonstrating the expression of CD34, and CD31 with flow cytometry.. Results： After ld in culture , shape changed cells were observed. After 2d in culture, large cell clusters appeared. After 3d in culture, around these clusters some adherent sells were observed. After incubation for 4 days, attached sells grew to short spindle-shaped and double positive staining for Dil-AC-LDL and UEA-1 can be observed under a fluorescent microscope. On the seventh day, FACS analysis , the adherent cells were positive for CD34 （14.13±2.79） % and CD31 （54.67±3.44） %. Conclusion： EPCs exist in the peripheral blood, and can be differentiated into mature endothelial cells （ECs） by the stimulation of VEGF, bFGF, IGF and EGF. EPCs can be anappropriate way of the angiogenesis therapy.
出处
《医药世界》
2007年第1期64-66,共3页
Medicine World
