脂质体介导血管内皮生长因子基因片段在犬骨髓基质干细胞中的表达及其影响  

Expression of vascular endothelial growth factor gene mediated by lipofectamin in canine bone marrow mesenchymal stem cells

作　　者：黄启顺[1] 李明静[1] 郑怀远[1] 韩阿超[1] 翁雨雄[1] 陈振兵[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院手外科,武汉430022

出　　处：《中华手外科杂志》2012年第3期173-176,共4页Chinese Journal of Hand Surgery

基　　金：湖北省自然科学基金资助项目（2007ABA116）

摘　　要：目的探讨脂质体介导的重组血管内皮生长因子165（vascular endothelial growth factor 165，VEGF165）基因转染后的犬骨髓基质干细胞分泌血管内皮生长因子（vascular endothelial growth factor，VEIGF）的能力及细胞增殖能力的改变，为改善骨缺血坏死的干细胞治疗方法提供基础。方法实验分VEGF165基因转染组与空白对照组，从成年犬的胫骨抽取骨髓，体外分离纯化，贴壁培养骨髓基质干细胞，传代培养后以脂质体法将携带荧光蛋白VEGF165基因的穿梭质粒转入实验组骨髓基质千细胞中，在荧光显微镜视下观察转染效率，并通过ELISA法分别检测转染后1、3．5、7、9、11、13d实验组与对照组培养液中VEGF的含量，对结果进行统计分析。MTT法检测转染细胞的增殖能力，绘制增殖曲线。结果重组人血管内皮生长因子165（recombinate the human being vasular endothelial growth factor165，hVEGF165）基因通过脂质体能够成功转染犬骨髓基质干细胞并分泌VEGF。h-VEGF165基因转染组培养液中VEGF蛋白含有量较未转染组增高，ELISA结果显示转染后上清液中第2天即可出现VEGF浓度的增高，到第7天时达到分泌高峰，筛选后的细胞上清液中VEGF浓度稳定在300ng／L左右，与对照组相比升高约10倍，其差别具有显著的统计学意义。骨髓基质干细胞转染基因后，细胞的增殖能力同对照组相比无明显差别。结论采用基因转染技术可将hVEGF165基因转染到犬骨髓间充质干细胞中并可有效表达VEGF。hVEGF165基因转染犬骨髓基质干细胞对细胞的增殖能力无明显影响。Objective To investigate the vascular endothelial growth factor （VEGF） secretion potential of canine bone marrow mesenchymal stem cells （BMSCs） that were tranfected with hVEGF165 gene, and to provide scientific basis for stem cell treatment in avascular bone necrosis. Methods Marrow stromal cells were obtained from the tibia of adult dogs. The cells were isolated and differential cultured in vitro to obtain BMSCs. An hVEGF165 gene transfection group and a control group were set up, In the gene transfection group after passage the recombinant PeDNA32hVEGF165 plasmid was transfected into the BMSCs with lipofectamin and the tranfection efficiency was observed under fluorescent microscope. For both groups VEGF165 expression levels in these BMSCs were quantified by ELISA at 1,3,5,7,9,11 and 13 days. MTT method was applied to detect cell proliferation and draw the growth curve. Results Canine BMSCs were successfully tmnsfected with hVEGF165 via lipofectamin and secreted VEGF proteins. VEGF secretion in culture media of hVEGF165 transfection group was higher than that of the control group （ P 〈 0.05）. ELISA detected increased VEGF secretion in 48 hours of canine BMSCs culture which peaked in 1 week in the hVEGF165 transfection group. VEGF concentration was around 300 ng/L, being 10 folds of that of the control group. The difference was significant （ P 〈 0.05）. BMSCs growth curve of the transfection group and control group was not significantly different （ P 〉 0.05）. Conclusion hVEGF165 can be tranferred into canine BMSCs using gene tranzfection technique. The tramfected BMSCs express VEGF protein of biological activity. Cell proliferation is not affected.

关 键 词：血管内皮生长因子 骨髓基质干细胞 脂质体 质粒 重组血管内皮生长因子165 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]