兔外周血内皮祖细胞的分离、培养和鉴定

Isolation, culture and identification of endothelial progenitor cells from rabbit peripheral blood

目的:研究新西兰大白兔外周血内皮祖细胞(EOCs)的分离、培养和鉴定方法,为后续实验研究做好准备。方法:通过耳中央动脉采集兔外周血,密度梯度离心法分离单个核细胞(MNCs),在EGM-2培养基的诱导分化下,获得EOCs。通过标记的乙酰化低密度脂蛋白(DiI-ac-LDL)摄取试验、荆豆凝集素(FITC-UEA-1)结合实验、CD34与VIII因子免疫组化染色、flk-1免疫荧光染色、体外血管形成实验以及细胞NO分泌功能测定,从细胞表面标记和细胞功能两方面对细胞进行鉴定。结果:新西兰大白兔外周血MNCs在体外培养可成功获得EOCs,EOCs能稳定摄取ac-LDL并与UEA-1结合,一致表达CD34、flk-1和VIII因子相关抗原。EOCs能分泌NO,并能在matrigel凝胶上形成稳定的血管腔样结构。结论:密度梯度离心法分离的兔外周血MNCs在一定的培养条件下能诱导分化成为EOCs。

Objective：To investigate how from rabbit peripheral blood as well as how to isolate and culture endothelial outgrowth cells （EOCs） to identify the cells in order to get ready for subsequent experiments. Methods：EOCs were obtained from in vitro culture of MNCs（Mononuclear cells） which were isolated with density gradient centrifugation from rabbit peripheral blood. The cells were identified by double positive for ac-LDL uptake and lectin binding, immunofluorescence of flk-1,immunocytochemistry of CD34 and Ⅷ factor related antigen,and then they were further identified by in vitro vasculogenesis activity and detection of NO secretion Results： EOCs could be obtained from in vitro culture of MNCs isolated from rabbit peripheral blood. EOCs were uniformly positive for ac-LDL uptake、lectin binding, and expressing flk-1 and Ⅷ factor related antigen. EOCs were capable of secreting NO and assembling into primitive vascular tube-like structures when plated in Matrigel. Conclusion： EOCs can be obtained from rabbit peripheral blood when a certain way of isolation and cultivation are performed.