慢病毒载体介导hHGF基因在大鼠真皮间充质干细胞的表达

目的探讨慢病毒载体（LVs）介导人肝细胞生长因子（hHGF）基因在大鼠真皮间充质干细胞（rdMSCs）的表达，以及hHGF基因修饰对rdMSCs增殖的影响。方法体外分离培养rdMSCs，hHGF—GFP—LV以不同感染复数（MOI）转染rdMSCs，荧光显微镜和流式细胞仪检测GFP阳性细胞比例；ELISA法测定感染后细胞上清hHGF水平；采用台盼蓝拒染法对hHGF基因修饰rdMSCs和rdMSCs进行细胞计数，绘制细胞生长曲线分析细胞的活力。结果rdMSCs是贴壁细胞，表达CD44和CD90，不表达CD34和CD45表面分子，可向脂肪细胞分化；MOI为100时，rdMScs能达到80％以上的感染效率；hHGF基因修饰rdMSCs细胞上清的hHGF水平高于阴性对照组（P〈0．05）。P4、P6和P8细胞的培养上清hHGF水平的差异无统计学意义（P〉0．05）；hHGF基因修饰rdMSCs有较强的增殖能力。其细胞生长曲线与rdMSCs相似。结论LVs可以介导hHGF基因在rdMSCs稳定表达，且不影响细胞的增殖活力。

Human hepatocyte growth factor gene expression in rat dermis mesenchymal stem cells by lentiviral vectors mediated gene transfer

Aim To investigate hHGF gene expression and reproductive activity of rdMSCs after lentiviral gene transfer. Methods rdMSCs were isolated in vitro. These Cells were transfeeted by hHGF-GFP-LVs at multiple MOI. Transfection efficiency was assayed by fluorescence microscope, rdMSCs were transfected by hHGF-GFP-LVs and NC-GFP- LVs at MOI of 100 respectively. After subculture,hHGF in cell supernatant was measured by ELISA assay. Growth curve analysis was used to evaluate the proliferation of rdMSC after genetic manipulation. Results rdMSCs were plasticadherent cells expressing CD44 and CD90 and lacking expression of CD34 and CD45 surface molecules. They could differentiate to adipocytes. A transduction efficiency more than 80% could be achieved at MOI of 100. rdMSCs tranfected with hHGF genes displayed a marked increase in the levels of hHGF compared with negative control（P〈0.05）. The difference in the means of hHGF among the fourth,sixth and eighth passage of cells was not statistically significant （P〉0.05）. The tranfected hHGF did not affect the proliferation of rdMSC. Conclusions rdMSC can persistently express hHGF by lentiviral transfection and maintain their reproductive activity in vitro.