VEGF165基因转染骨髓源性血管内皮祖细胞的体外实验研究

An in vitro study of PcDNA3.0-hVEGF165 transfection on endothelial progenitor cell derived from murine bone marrow

目的探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)转染骨髓源性血管内皮祖细胞(endothelial progenitor cell,EPC)后,EPC 分泌 VEGF 及活性以及 EPC 特性的影响。方法体外获取大鼠骨髓,分离单个核细胞,经培养,鉴定为 EPC。用脂质体介导 pcDNA3.0-hVEGF165转染,转染后 ELISA 法测培养上清 VEGF 蛋白水平,培养上清液对 ECV304细胞生长的影响,以评价 VEGF 蛋白活性,MTF 法评价转染对细胞活性的影响,免疫细胞化学检测 EPC 表达 VEGF、vWF(血友病因子)、VEGF-R2(血管内皮生长因子受体2)的变化。结果转染后的第7天表达的VEGF 蛋白浓度达1280 pg/ml,转染对 EPC 的增殖无影响,表达 VEGF、vWF、VEGF-R2的比率随时间而逐渐升高,至第7天时分别为88.52%、82.65%和95.97%。结论 VEGF 转染的 EPC 能表达一定浓度有功能的 VEGF 蛋白,能帮助 EPC 保持内皮细胞特性,向成熟内皮细胞分化。

Objective To investigate the effect of VEGF gene transfection on endothelial progenitor cell derived from murine bone marrow. Methods Wistar rat＇s bone marrow was obtained, mononuclear cell isolated, and endothelial progenitor cells （EPS） were cultured in EGM-2MV. EPCs were identified by immunocytochemistry and electron microscope. EPCs were transfected by liposome mediated pcDNA3.0-hVEGF165. VEGF protein level was determined in the cultural medium supernatant after VEGF transfection by ELISA. Cultural medium supernatant was used to co-culture with ECV304, VEGF protein activity was evaluated by MTT. EPCs expression of vWF, VEGF, FLK-1 was detected by immunocytochemistry. Results EPCs were effectively enriched by EGM-2MV, and the EPCs obtained express the typical cell surface markers such as CD34, CD133, FLK-1. The concentration of VEGF protein in supernatant reaches 1280 pg/ml in the 7th day after pcDNA3.0-hVEGF transfection. No influence of EPCs proliferation could be found after transfection. The cell surface marker expression of VEGF, FLK-1, vWF became higher with time, and the ratios of positive cell were 88.52%, 82.65% and 95.97% respectively. Conclusions pcDNA3.0-hVEGF165 transfect EPCS mediated by liposome could excrete a high concentration of functional VEGF protein. It is helpful for EPC to maintain the characters of endothelial cell after VEGF gene transfection and differentiate to mature endothelial cell.