摘要
目的探索外周血内皮祖细胞的诱导培养方法。方法用密度梯度离心分离健康志愿者外周血单个核细胞,EGM-2培养基重悬并在纤连蛋白包被的培养板中进行诱导培养,动态观察贴壁细胞生长状况,免疫荧光技术鉴定内皮祖细胞特性,流式细胞术检测细胞周期分布及其相关表面标志CD34、CD133、CD31、VEGFR2和CD14。结果贴壁细胞呈细长条状分布,整体形态和生长密度均以第9天最佳,约占外周血单个核细胞的4.62%～5.47%;细胞停留在G0/G1期,增殖指数仅为1.20%±0.18%;细胞自第5天起即可同时吞噬乙酰化低密度脂蛋白和结合荆豆类凝集素,Ⅷ因子相关抗原于第9天始呈阳性;表面标志CD34、CD133、CD31、VEGFR2和CD14分别为0.19%±0.06%、1.67%±0.52%、61.56%±5.57%、70.29%±7.37%和89.31%±4.11%,共同指示诱导所得细胞属于正在分化的内皮祖细胞。结论在特定条件下可直接从外周血单个核细胞中诱导培养出内皮祖细胞。
Objective To investigate induction of endothelial progenitor cell（EPC）from peripheral blood.MethodsPeripheral blood mononuclear cells（PBMCs） were isolated from healthy volunteers with density gradient centrifugation.Cells were suspended in EGM-2 medium and cultured in the 6-well plate which was coated with fibronectin.During cultivation,adherent cells were kept on observing and then examined by immunofluorescence staining for characteristic while flow cytometry for cell cycle and surface markers such as CD34,CD133,CD31,VEGFR2 and CD14.Results Adherent cells turned slenderness gradually,accounting for 4.62%～5.47% of PBMCs.Almost all of them were arrested at G0/G1 stage,with a proliferation index about 1.20%±0.18%.Cells began to uptake acLDL and exhibit lectin binding capability simultaneously since the fifth day and then form enough Ⅷ factor related antigen on day 9.The expression of EPC related surface markers including CD34,CD133,CD31,VEGFR2,CD14 were 0.19%±0.06%,1.67%±0.52%,61.56%±5.57%,70.29%±7.37% and 89.31%±4.11% respectively.All these indicated that adhesion cells were differentiating EPC.Conclusion Under specific condition,it is possible to induce EPC from PBMCs directly.
出处
《基础医学与临床》
CSCD
北大核心
2010年第7期743-748,共6页
Basic and Clinical Medicine
基金
广东省自然科学基金(07300312)
国防科工委专项课题(B3320062101)
关键词
外周血单个核细胞
内皮祖细胞
诱导
peripheral blood mononuclear cells
endothelial progenitor cell
induction
