应用转基因技术体外培养表达人碱性成纤维细胞生长因子的视网膜色素上皮细胞

目的 探讨应用转基因技术体外培养稳定表达人碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)的视网膜色素上皮(retinal pigment epithelium,RPE)细胞的可行性。方法 构建以绿色荧光蛋白(green fluorescent protein,GFP)作为标记基因的人bFGF真核表达载体pcFG。应用脂质体介导法转染人RPE细胞,以G418筛选出表达bFGF的RPE细胞,并进行细胞克隆,传代培养4周。于荧光显微镜下观察GFP的表达情况,用原位杂交和免疫组织化学染色法检测bFGF在RPE细胞的表达情况。结果 酶切结果证实含有GFP的真核表达载体pcFG构建正确。在荧光显微镜下可见RPE细胞表达绿色荧光蛋白。经原位杂交和免疫组织化学染色证实,转染pcFG后的RPE细胞内有大量bFGF—mRNA的转录蛋白表达。结论 应用转基因技术可体外培养稳定表达bFGF的RPE细胞。

Stable expression of human basic fibroblast growth factor gene in retinal pigment epithelial cells by gene transfer procedures

Objective The purpose of the present study was to explore whether gene transferred retinal pigment epithelial cells (RPE) can constantly express human basic fibroblast growth factor gene (hbFGF) and to study the feasibility of using this procedure for the treatment of retinitis pigmentosa. Methods Plasmid carrying hbFGF [using green florescent protein (GFP) as a reporter gene] was constructed and transferred into the RPE cells by lipofectamine. The positively expressed cell clones were selected with G418 and cultured for 4 weeks. Expression of GFP gene was identified by the fluorescence microscope. Expression of hbFGF in the RPE cells was determined by in situ hybridization and immunohistochemical methods. Results Reconstruction enzyme digestion demonstrated that the eukaryotic expression pcFG was constructed correctly. Expression of GFP protein in the gene transferred RPE was detected under the fluorescence microscope. Enzyme digestion and agarose gel electrophoresis analysis showed that the gene transferred RPE expressed the hbFGF gene. Conclusions RPE cells are able to express hbFGF gene stably by gene transfer procedures.