兔外周血两种内皮祖细胞的分离培养和鉴定

目的研究新西兰大白兔外周血两种内皮祖细胞(EPC和EOC)的分离培养和鉴定。方法由兔耳中央动脉采集外周血,以密度梯度离心法分离单个核细胞(MNC),置于EGM-2培养基中培养。用DiI标记的乙酰化低密度脂蛋白(DiI-acLDL)摄取试验和FITC标记的荆豆凝集素(FITC-UEA-1)结合试验,检测flk-1表达的免疫荧光法和检测CD34、Ⅷ因子表达的免疫组化染色法,以及体外血管形成试验对EPC/EOC进行鉴定。结果从新西兰大白兔外周血MNC中可成功地培养出EPC和EOC。培养第7天的EPC和第16天的EOC均能吞噬ac-LDL并与凝集素UEA-1相结合,同时均可表达CD34、flk-1和Ⅷ因子相关抗原。EOC在matrigel凝胶上可形成血管腔样的结构。结论用密度梯度离心法从兔外周血中分离的MNC在一定的培养条件下,能分化成为EPC和EOC,为后续的实验研究提供了细胞来源。

Isolation, culture and identification of two types of endothelial progenitor cells from peripheral blood in rabbits

AIM: To investigate how to isolate, culture and identify two types of endothelial progenitor cells from peripheral blood in rabbits. METHODS: Mononuclear cells(MNCs) were isolated from rabbit peripheral blood. Endothelial progenitor cells (EPCs) and endothelial outgrowth cells (EOCs) were obtained from MNCs through different ways of isolation and culture. Two types of cells were assessed by DiI-ac-LDL uptake and lectin binding, and then they were identified by immunofluorescence of flk-1, immunocytochemistry of CD34 and VIII factor related antigen and vasculogenesis activity in vitro. RESULTS: Two types of endothelial progenitor cells were obtained from rabbit peripheral blood through different ways of isolation and culture. EPCs on the seventh day and EOCs on the sixteenth day were positive for ac-LDL uptake and lectin binding, and both of them expressed CD34, flk-1 and VIII factor related antigen. EOCs were assembled into primitive vascular tube-like structures when plated in matrigel. CONCLUSION: EPCs and EOCs could be obtained from rabbit peripheral blood when different ways of isolation and culture were performed. The system of cell culture can be applied to subsequent experiments in cell transplantation.