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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。
Objective To investigate the expression of human hepatocyte growth factor (hHGF) gene in rat dermal mesenchymal stem cells (rdMSCs) mediated by lentiviral vector (LVs) and the effect of hHGF gene modification on the proliferation of rdMSCs. Methods The rdMSCs were isolated and cultured in vitro. The rHMSCs were transfected with hHGF-GFP-LV at different MOI. The proportion of GFP positive cells was detected by fluorescence microscopy and flow cytometry. The hHGF levels in the supernatant of the cells were detected by ELISA. Anti-dye method of hHGF gene modified rdMSCs and rdMSCs were counted, and cell growth curve was drawn to analyze cell viability. Results rdMSCs were adherent cells expressing CD44 and CD90, but not expressing CD34 and CD45 surface molecules, which could differentiate into adipocytes. When the MOI was 100, rdMSCs could reach more than 80% infection efficiency. HHGF gene modified rdMSCs supernatant hHGF The level was higher than the negative control group (P <0.05). There was no significant difference in the level of hHGF in the culture supernatants of P4, P6 and P8 cells (P> 0.05). The hHGF gene-modified rdMSCs had strong proliferative capacity and the cell growth curve was similar to rdMSCs. Conclusion LVs can mediate the stable expression of hHGF gene in rdMSCs without affecting the proliferation activity of cells.