VEGF基因体外转染大鼠骨髓间充质干细胞的实验研究

目的:探讨脂质体介导血管内皮细胞生长因子(VEGF)基因转染大鼠骨髓间充质干细胞(MSCS)应用于基因治疗的可行性、安全性。方法:体外分离、培养、鉴定MSCs,PcDNA3.1(-)/VEGF165质粒转染MSCs,转染后用免疫荧光和ELISA检测MSCs表达VEGF蛋白的情况,MTT检测MSCs对VEGF质粒转染的敏感性。结果:骨髓中分离得到MSCs,流式细胞检测显示MSCs不表达CD34和CD45,但表达CD90。透射电镜观察可见细胞浆中含大量粗面内质网和分泌颗粒。VEGF基因转染MSCs后第5天抗VEGF免疫荧光染色约90%的MSCs呈阳性,ELISA检测结果显示PcDNA3.1(-)/VEGF165质粒转染组细胞培养上清液中VEGF含量明显高于对照组,并于转染后第5天达到峰值。MTT检测结果显示VEGF质粒转染对MSCs增殖无影响。结论:MSC可作为VEGF基因转染的靶细胞用于基因治疗。

Study of VEGF gene transduction on rat mesenchymal stem cells in vitro

Objective To investigate the feasible and security of Vascular endothelial growth factor (VEGF) transduction on rat mesenchymal stem cells (MSCs) mediated by liposome. Methods MSCs were isolated from Sprague-Dawley (SD) rat bone marrow and cultured in vitro. Cultured MSCs were analyzed by fluorescence-activated cell sorting (FACS). Plasmid PcDNA3.1(-)/VEGF165 containing VEGF gene was transduced into the MSCs by liposome. MSCs without transduction were used as controls. Human VEGF expression in vitro was assessed by immunofluorescence staining and ELISA. MTT method was used to detect the effect of transduction on MSCs. Results The MSCs obtained from rats bone marrow did not express CD34 and CD45,however,almost all the cells expressed CD90. Approximately 90% of cells in VEGF-transduced MSCs group were stained positively on day 5 after gene transduction. There was no VEGF expression in non-transduced MSCs group.The level of human VEGF in the culture medium of VEGF-transduced MSCs was significantly increased and reached a peak at day 5,and no VEGF was detected in the culture medium of non-transduced MSCs. It wasn't found that VEGF transduction had a significantly inhibitory effect on MSC. Conclusion MSC can be target cell for VEGF transduction,and provide a novel gene therapentic strategy for neovascularization which can possibly apply for clinic usage.