腺病毒介导人内皮抑素感染视网膜色素上皮细胞的可行性研究  

The feasibility of an adenovirus-mediated delivery of human endostatin in cultured human retinal pigment epithelial cells

作　　者：张美霞[1] 吴静[1] 辛梅[1] 张军军[1] 闫乃红[1] 严密[1] 

机构地区：[1]四川大学华西医院眼科,四川成都610041

出　　处：《眼视光学杂志》2009年第4期265-268,共4页Chinese Journal of Optometry & Ophthalmology

基　　金：国家自然科学基金项目(30500547);教育部重点项目(108178)

摘　　要：目的探讨以腺病毒为载体对体外培养的人视网膜色素上皮(human retinal pigment epithelial,hRPE)细胞进行人内皮抑素(human endostatin,hES)感染,获得高效表达hES转基因细胞的可行性。方法常规培养hRPE细胞,传代至第3～第5代后,应用携带有绿色荧光蛋白(green fluorescent protein,GFP)的hES重组腺病毒按感染复数30进行感染。培养24h后,在荧光显微镜下计数培养的hRPE细胞的GFP表达阳性率;用免疫组织化学法观察hES在已感染hRPE细胞中的表达情况;提取hRPE细胞总RNA,采用RT-PCR技术对产物行琼脂糖凝胶电泳;用Western blot法检测感染hRPE细胞培养上清液中hES的表达水平。结果hES重组腺病毒感染后,hRPE细胞形态正常,感染后24h,GFP即呈100%表达,弥漫整个胞质;免疫组织化学法观察可见,hRPE细胞浆内呈棕黄色的阳性反应,而在感染空载腺病毒的对照组中细胞胞浆无染色;RT-PCR反应可见,细胞裂解产物中有hES阳性条带;Western blot法检测结果表明,培养上清液中有hES蛋白的表达。结论hES重组腺病毒载体能有效感染体外培养的hRPE细胞,并稳定表达hES蛋白。Objective To obtain transgenic human retinal pigment epithelial （hRPE） cells by using an adenovirus-mediated delivery of the eDNA of human endostatin （hES）. Methods The hRPE cells were cultured in the usual way, and the cells were then infected by two passages using a recombinant hES adenovirns vector at a dosage of MOI （multiplicity of infection） 30. The expression of GFP in hRPE cells was examined using a fluorescence microscope at 24 h after infection. The production of hES mRNA and protein was evaluated by RT-PCR and immunohistochemistry. The expression of the hES protein in the supernatant fluid of infected hRPE cells was detected by Western blot. Results GFP expression in hRPE cells could be detected 24 b after infection and the positive ratio was almost 100%. Immunohistochemistry showed all hRPE cells expressing the hES protein. The RT-PCR result was detected in a specific band with a length of about 623 bp in the genomic DNA. Western blot showed hES protein expression in the supernatant fluid of infected hRPE cells. Conclusion The recombinant hES adenovirns vector can effectively infect cultured hRPE cells and the infected cells show a high expression of hES.

关 键 词：视网膜色素 上皮细胞 腺病毒 内皮抑素类 感染 

分 类 号：R774.1[医药卫生—眼科]