人内皮抑素重组腺病毒的构建及其在人视网膜色素上皮细胞的表达

Construction of recombinant human endostatin adenovirus vector and its expression in human retinal pigment epithelial cells

目的观察构建的含人内皮抑素(human endostatn,hES)基因腺病毒载体对人视网膜色素上皮(human retinal pigment epitheliaum,hRPE)细胞的感染及其基因表达情况,为眼内新生血管性疾病治疗提供一定的实验依据。方法原代培养hRPE细胞,行角蛋白免疫组织化学进行鉴定。从真核表达载体pEGFP-N1-ASP-hES将ASP-hES扩增,亚克隆至pAdTrack CMV穿梭质粒中,与pAdEasy1质粒在大肠杆菌BJ5183中进行同源重组为腺病毒载体,线性化后转染293细胞进行包装扩增。用该腺病毒感染hRPE细胞,应用荧光显微镜和Western blot检测病毒感染及外源基因的表达情况。结果角蛋白免疫组织化学显示所培养的细胞为hRPE细胞,感染24h后荧光显微镜下可见RPE细胞中有大量的绿色荧光蛋白表达,Western blot结果显示hES蛋白高表达。结论构建的hES腺病毒对hRPE具有极高的感染效率,可作为一种良好的基因导入载体,为ES基因治疗眼内新生血管性疾病的进一步研究奠定了基础。

Objective To investigate the interference and expression of human endostatin（hES） recombinant adenovirus vector on human retinal pigment epithelial（RPE） cells,and to evaluate the roles of hES in the neovascular ocular diseases. Methods Human RPE cells （hRPE） were primary cultured and identified with keratin immunohistochemical method. ASP-hES sequence from the eukaryotic expression vector pEGFP-N1-ASP-hES with PCR was ampligied and subcloned into the pAdTrack CMV shuttle vector. The resultant plasmid （pAdTrack CMV ASP-hES） was co-transduced into E. coli BJ5183 cells with pAdEasy 1 plasmid to undergo homologous recombination. The linearized recombinant plasmid was transfected into 293 cells. Then the recombinant adenovirus vector was used to infect hRPE cells,the infection and expression of gene were tested under fluorescent microscope examination and Western blot analysis. Results The hRPE cells were identified by keratin immunohistochemica I staining. A great quantity of green fluorescent protein and the expression of hES were observed in hRPE cells after 24 hours infected. The Western blot analysis indicated the high expression of hES. Conclusion The recombinant adenovirus Ad-ASP-hES is successfully constructed and the infective efficiency of adenovirus is greatly acceptable to hRPE cells, which is useful for studying angiogenesis gene therapy by ES genes.