大鼠骨髓源内皮祖细胞分离和培养

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

目的:研究大鼠骨髓源内皮祖细胞(EPCs)分离、培养以及诱导向内皮细胞分化的方法.方法:采用密度梯度离心法从大鼠骨髓中分离单个核细胞,经贴壁后定向诱导培养2周.以CD34、CD133、VEGFR-2、vWF免疫细胞化学、荧光染色法以及Dil-acLDL结合FITC-UEA-1双荧光染色法鉴定EPCs.结果:贴壁细胞经培养诱导后3d开始伸展,4～7 d形成细胞集落,10～21 d增殖加速呈典型的'鹅卵石'样外观,并出现条索状结构且呈'微血管样'排列生长.细胞免疫荧光染色鉴定CD34、CD133、VEGFR-2、vWF阳性,Dil-acLDL联合FITC-UEA-1双染阳性.结论:从大鼠骨髓中分离单个核细胞,体外诱导培养后可以表现出部分内皮细胞的特征.

Objective To investigate the methods of isolating and culturing endothelial progenitor cells(EPCs) from rat bone marrow,and to explore the differentiation conditions of EPCs into endothelial cells.Methods The mononuclear cells were isolated from rat bone marrow by using density-gradient centrifugation,then the adherent cells were orientation induced for 2 weeks.EPCs were identified with Dil-acLDL combined FITC-UEA-1 double fluorescence,and the expression of CD34,CD133,Flk-1 and vWF were also analyzed by immuocytochemistry and immunofluorescence.Results The adherent cells stretched at 3rd day after inducing culture,endothelial cell colony-forming units(CFU-ECs) and endothelial colony-forming cells(ECFCs) appeared at the 4th～7th day,the proliferation was increased and showed typical 'cobblestone' appearance at 10th～21st d,the cord-like structure were observed and grew as 'capillary' arrangement.CD34,CD133,Flk-1 and vWF of adherent cells were postively expressed in immunofluorescence and Dil-acLDL combined FITC-UEA-1 double fluorescence was also positive.Conclusion The mononuclear cells isolated from rat bone marrow could show partial characteristics of endothelial cells after inducing culture in vitro.