强力霉素诱导携带人血管内皮生长因子A-shRNA的1/2型重组腺相关病毒的包装及鉴定  

Package and identification of doxycycline-regulated recombinant adeno-associated virus serotype 1/2 encoding human vascular endothelial growth factor A-shRNA

作　　者：秦秀虹[1] 范松涛[1] 卢建民[1] 张珍珍[2] 

机构地区：[1]大连医科大学附属第一医院眼科,辽宁省大连市116011 [2]上海交通大学附属上海市第一人民医院眼科,上海市200080

出　　处：《眼科新进展》2013年第3期224-228,共5页Recent Advances in Ophthalmology

基　　金：辽宁省教育厅高等学校科研项目(编号:L2010127)~~

摘　　要：目的通过Helper-Free腺相关病毒包装系统包装由强力霉素(doxycycline,DOX)诱导的携带hVEGFA-shRNA-GFP的1/2型重组腺相关病毒(recombinant adeno-associated virus,rAAV),测定病毒滴度并检测其对人视网膜血管内皮细胞(human ret-inal vascular endothelial cell,HRVEC)血管内皮生长因子A(vascular endothelial growth factor,VEGFA)的敲除作用。方法将AAV-293细胞复苏、培养后传代。用磷酸钙转染法将质粒pAAV-hVEGFA-shRNA-GFP、1/2型腺相关病毒质粒(pAAV1/2)和pHelper质粒共转染AAV-293细胞,包装生成带有hVEGFA-shRNA-GFP的1/2型rAAV,将此rAAV感染AAV-293细胞,荧光显微镜观察病毒转染和感染效率。斑点杂交法测定病毒滴度。将HRVEC分为3组培养,正常对照组、HRVEC+rAAV组及HRVEC+rAAV+DOX诱导组,Western-blotting法检测VEGFA的敲除作用。结果 3种质粒共转染AAV-293细胞,发现病毒包装过程中细胞培养基颜色由红色变为橘黄色,细胞变圆并脱离培养皿底部,漂浮于培养基中。荧光显微镜下观察可见,24h后AAV-293细胞表达绿色荧光蛋白(green fluorescence protein,GFP),72h表达GFP细胞数比例可达95%～100%;共转染成功,包装得到DOX诱导的携带有hVEGFA-shRNA-GFP的1/2型rAAV,斑点杂交法检测病毒滴度为1015v.g.·L-1。HRVEC于重组病毒感染后第3天高表达GFP。免疫印迹法检测正常对照组、HRVEC+rAAV组及HRVEC+rAAV+DOX诱导组HRVEC中VEGFA表达量的变化发现:HRVEC+rAAV组其VEGFA表达量较正常对照组明显下降,HRVEC+rAAV+DOX诱导组其VEG-FA表达量较HRVEC+rAAV组明显降低。提示rAAV对细胞内VEGFA具有显著的敲除作用,并且DOX有加强VEGFA敲除的作用,病毒包装成功。结论 DOX诱导的携带有hVEGFA-shRNA-GFP的1/2型rAAV的包装获得成功,收获病毒具有较高滴度,能成功地感染HRVEC,并对HRVEC的VEGFA具有明显敲除作用,同时DOX有加强VEGFA敲除的作用,为眼底新生血管疾病基因治疗的研究提供了实验基础。Objective Through packaging doxycycline （DOX）-regulated recom- binant adeno-associated virus serotype 1/2 encoding human vascular endothelial growth factor A-shRNA （hVEGFA-shRNA） by AAV Helper Free system,to measure the virus ti- ter and detect the vascular endothelial growth factor A （VEGFA） knockdown effects in human retinal vascular endothelial cells （HRVEC）. Methods AAV-293 cells were re- covered, cultured and passaged. The plasmid of pAAV-hVEGF-shRNA-GFP, pPAAV1/2 and pHelper were cotransfected into AAV-293 cells by calcium acid phosphate method, and recombinant adeno-associated virus （rAAV） serotype 1/2 encoding hVEGF-shRNA was packaged to infect AAV-293 cells. Transfected and infected efficiency were meas- ured by fluorescence microscope, and virus titer was measured by dot-blot assay after rAAV was collected. HRVEC were divided into normal group, rAAV infected group and rAAV infected group with DOX, Western Blot method was used to detect the knockdown effects of VEGFA in HRVEC. Results AAV-293 cells were cultured after cotransfec- tion with three plasmids. The most obvious sign of viral production was a color change in the medium from red to orange or yellow, and some cells rounded up and detached from the plate with floating in the medium. AAV-293 cells expressed green fluorescence protein （GFP） at 24 hours after three plasmids cotransfection under fluorescence mi- croscope and the proportion of cells expressing GFP reached from 95% to 100% at 72 hours after cotransfection,which indicated the succeed of the cotransfection, the pack- age rAAV that encoding hVEGFA-shRNA was succeeded and the virus titer was 1 × 10^15 v.g. L^-1 measured by dot-blot assay. HRVEC highly expressed GFP at 3 days after rAAV infection. VEGFA expression reduced significantly in rAAV infected group com- pared to control group, and VEGFA expression reduced significantly in rAAV infectedgroup with DOX compared to rAAV infected group tested by Western blot method, which indicated that rAAV could knock- down V

关 键 词：重组腺相关病毒 SHRNA 强力霉素 血管内皮生长因子 

分 类 号：R331.2[医药卫生—人体生理学]