改构aFGF在角膜内皮细胞中的分泌表达及意义

Secretary expression of modified human aFGF in corneal endothelial cell and its significance

目的观察改构型人酸性成纤维细胞生长因子(m-haFGF)真核表达载体(pSecTag/m-haFGF)在培养的兔角膜内皮细胞中的分泌表达,探讨其表达产物对由生理盐水损伤的角膜内皮细胞的保护作用。方法采用脂质体转染的方法,将psecTag/m-haFGF转染培养的兔角膜内皮细胞,用免疫印迹法检测m-haFGF的表达情况,同时用生理盐水造成细胞损伤,用MTT法检测细胞存活状况。结果角膜内皮细胞在转染m-haFGF基因后能够表达目标蛋白,未转染组为阴性;MTT显示,转染后损伤的细胞存活状况明显好于未转染组(P<0.05)。结论转染pSecTag/m-haFGF能够在培养的兔角膜内皮细胞中分泌表达,其基因表达产物对生理盐水造成的角膜内皮细胞损伤具有保护作用。

Objective To observe the secretary expression of constructed eukaryotic expressive vector of modified human acidic fibroblast growth factor ( m-haFGF) in cultured rabbit corneal endothelial cells, and evaluate its protection on corneal endothelial cells injured by 0. 9% sodium chloride solution. Method Plasmid vector of pSecTag/m-haFGF was transferred into the rabbit corneal endothelial cells by the liposome-mediated method, and expression of m-haFGF was tested using western blotting after 48 hours. Corneal endothelial cells of rabbits were exposed to 0. 9% sodium chloride solution to create cell injury, and the cellular viability was evaluated using MTT assay. Results After transfer 24 hours,the results of western blotting were positive in the liposome pSecTag/m-haFGF group and were negative in the control group. The cellular viability was significantly enhanced in the m-haFGF transferred group in comparison with the control group (P < 0. 05). The spontaneous beat rate in the m-haFGF transferred group was significantly higher than that in control group ( P < 0. 05 ). Conclusion The pSecTag/m haFGF eukaryotic vector can be successfully expressed and pSecTag/m-haFGF have a protective effect on corneal endothelial cells injured by 0. 9% sodium chloride solution.