鼠骨髓源性内皮祖细胞的分离培养与鉴定

Isolation,Culture and Identification of Rat Bone Marrow-Derived Endothelial Progenitor Cells

目的探讨大鼠骨髓源性内皮祖细胞(endothelial progenitor cells,EPCs)的分离培养鉴定的方法。方法Percoll(1.077g/ml)分离液分离大鼠骨髓单个核细胞,血管内皮生长因子(Vascular Endothelial Growth Factors,VEGF)和碱性成纤维细胞生长因子(basic Fibroblast Growth Factors,bFGF),对其进行诱导培养,光镜观察EPCs形态,免疫荧光检测血小板内皮细胞粘附分子-1(PECAM-1/CD31)、血管内皮钙粘蛋白(VEcadherin/CD144)、荆豆凝集素-1(FITC-UEA-1)的表达和摄取Dil荧光标记的乙酰化-低密度脂蛋白(Dil-ac-LDL)。结果诱导培养7d后,可见集落和铺路石样结构,激光扫描共聚焦显微镜(Laser Scanning Confocal Microscope,LSCM)显示表型为CD31+VE-cadherin+双阳性细胞以及具有内皮细胞功能的Dil-ac-LDL和FITC-UEA-1双染色细胞。结论采用Percoll(1.077g/ml)密度梯度离心结合VEGF、bFGF诱导培养可以获得EPCs,说明该培养方法可行。

Objective To study the methods of isolation, culture and identification of endothelial progenitor ceils （EPCs） from rat bone marrow. Methods Mononuclear eells suspension of rat bone marrow was prepared by density gradient centrifugation on Percoll （ 1. 077 g/ml）. The mononuclear cells were induced by VEGF and bFGF, and then the shape of these cells were observed by light microscopy. EPCs were identified by staining for the expression of PECAM- 1/CD31, VE-cadherin/CD144, fluorescein Ulex Europaeus agglutinin-1 （FITC-UEA-1） and uptake of DiI complexed acetylated low-density lipoprotein （DiI-ac-LDL）. Results The clone-like morphology and the “cobblestone” structure were observed by inverted microscopy after 7 days cultivation. Laser Scanning Confocal Microscopy （LSCM） showed that the adherent ceils were positive for CD31 and VE-cadherin. Moreover the cells could incorporate Dil-ac-LDL and bind FITC- UEA-1. Conclusion 1. 077 g/ml percoll density centrifugation, with VEGF and bFGF induction may obtain EPCs from rat bone marrow, thus demonstrating the feasibility of the methods used.