大鼠骨髓源性血管内皮祖细胞体外培养分化及鉴定

The culture in vitro,differentiation and identify of murine bone marrow-derived endothelial progenitor cells

目的:探索大鼠骨髓源性血管内皮祖细胞(EPCs)的培养、诱导分化及鉴定方法。方法:冲洗大鼠骨髓腔,梯度密度离心法获得单个核细胞,内皮细胞培养液EGM-2MV培养,通过细胞形态,免疫组化和免疫荧光检测CD34、VEGFR-2、vWF,CD133,摄取Dil-ac-LDL和结合FITC-Lectin-UEA-1,超微结构及染色体核型分析进行鉴定。结果:新分离的骨髓单个核细胞呈圆形,大小不一;48h后部分细胞开始贴壁,呈梭形、纺锤形或不规则形;4～8d呈细胞集落或线状排列;9～11d接近融合,呈典型鹅卵石样外观。贴壁细胞CD34、CD31、VEGFR-2、vWF、CD133均表达阳性并呈动态变化,能够摄取Dil-ac-LDL和结合FITC-Lectin-UEA-1,透射电镜见特征性Weible-Palade小体,能稳定保持二倍体核型。结论:本实验初步建立了一套大鼠骨髓血管内皮祖细胞分离、培养、诱导分化及鉴定方法体系。

Objective： To explore the procedures of culture, differentiation and identify of murine marrow-derived endothelial progenitor cells （EPCs）. Methods： The bone marrow mononuclear cells （BMMNCs） were gained by washing cavitas medullaris of rats and then the washings being accepted density gradient centrifugation, and cultured in endothelial growth medium-2. These cells were identified by cellular morphology, expressions of CD34, VEGFR-2, vWF and CD133, uptaking Dil-ac-LDL, combining with FITC-Lectin-UEA-1, cellular uhrastructure, karyotype analysis on chromosome. Results： The fresh separated BMMNCs displayed as sphericity and their sizes were different. Some cells started adhering to culture plates 48 hours after planting, and present as fusiform and irregular shape. On the 4th -8th day of planting, cells formed colonies or lined up. Up to the 9th -11th day, the cells approached confluens and exhibited cobblestone-like appearance. The attached cells expressed CD34, CD31, VEGFR-2, vWF and CD133 antigens, could take in Dil-ac-LDL and combine with FITC-Lectin-UEA-1. The characteristic Weible-Palade bodies were observed by transmission electron microscope （TEM）, which could maintain cellular diploid nucleus. Conclusion： The system including isola- tion, culture, differentiation and identify of murine marrow-derived EPCs was primitively established.