腺相关病毒介导bFGF基因对虹膜色素上皮细胞的转染及表达

Expression of bFGF gene in iris pigment epithelial cells tranfected adeno-associated virus-mediated basic fibroblast growth factor gene

目的:评价重组腺相关病毒(rAAV)载体介导碱性成纤维细胞生长因子(bFGF)基因转染体外培养的虹膜色素上皮细胞(IPE)后,bFGF基因在细胞内转录和翻译水平的表达情况。方法:取IPE细胞,转染含有分泌型bFGF全长cDNA和巨细胞病毒启动子的rAAV-bFGF,以未转染和同样条件下转染rAAV-GFP的细胞作对照。1周后,RT-PCR检测细胞中bFGFmRNA的表达。ELISA法检测细胞中bFGF蛋白的表达。结果:转染rAAV-bFGF的IPE细胞有bFGFmRNA的表达,转染rAAV-GFP和未转染细胞无bFGFmRNA的表达。以105个细胞计算,转染rAAV-bFGF的IPE细胞bFGF蛋白含量为(1.322±0.663)μg/L,高于未转染组的(0.213±0.036)μg/L和转染rAAV-GFP组的(0.243±0.050)μg/L,差异有统计学意义(F=18.294,P=0.000)。结论:IPE细胞能被rAAV-bFGF转染,转染细胞能够在转录和翻译水平表达bFGF基因。

Aim： To determine whether iris pigment epithelial （IPE） cells could be transfered by recombinant adenoassociated virus （rAAV） mediated basic fibroblast growth factor （bFGF） gene and detect the expression of bFGF gene. Methods：lPE cells were allocated into two groups, and transfected with rAAV-bFGF and rAAV-GFP, respectively, then were cultured for 1 week. bFGF mRNA and protein of transfected and untransfected cells were analyzed by RT-PCR and ELISA method, respectively. Results：bFGF gene mRNA was detected in cells transfected with rAAV-bFG,but not in cells transfected with rAAV-GFP and untransfected cells, bFGF protein expression in the 105 cells transfected with rAAVobFGF was （ 1. 322 ±0.663） μg/L, which was higher than those of （0. 213 ±0.036） μg/L in untransfected cells and（O. 243 ± O. 050） μg/L in cells transfected with rAAV-GFP（ F = 18. 294 ,P = O. 000）. Conclusion ： IPE cells can be transfected with rAAV-bFGF, and transfected cells can secret bFGF protein.