腺相关病毒介导色素上皮衍生因子转染人虹膜色素上皮细胞的体外研究  

Adeno-associated virus mediated transduction of pigment epithelium-derived factor to human iris pigment epithelial cells in vitro

作　　者：赵莼[1,2] 王方[2] 计菁[1] 施沃栋[1] 范先群[1] 罗敏[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院眼科,上海200011 [2]同济大学医学院附属第十人民医院眼科,上海200072

出　　处：《上海交通大学学报（医学版）》2011年第8期1078-1081,共4页Journal of Shanghai Jiao tong University:Medical Science

基　　金：上海市卫生局基金(2006028)~~

摘　　要：目的探讨以腺相关病毒(AAV)为载体,对体外培养的人虹膜色素上皮(IPE)细胞进行色素上皮衍生因子(PEDF)转染,获得高效表达PEDF转基因细胞的可行性。方法构建携带PEDF基因的重组AAV载体AAV-PEDF,体外培养人IPE,用1×106pfu的AAV-PEDF按感染复数(MO I)为0、2、10和20分别感染体外培养的IPE细胞;荧光显微镜下观察细胞绿色荧光蛋白表达情况;流式细胞术检测细胞转染效率;RT-PCR和W estern b lotting法鉴定PEDF在IPE细胞中的表达。结果 AAV-PEDF转染后,IPE细胞生长正常,用荧光显微镜、RT-PCR和W estern b lotting均检测到PEDF表达。在MO I为20时,细胞感染阳性率达64.22%。结论 AAV-PEDF能有效地转染体外培养的IPE细胞并有效表达PEDF。Objective To investigate the feasibility of recombinant adeno-associated virus(AAV) mediated transduction of pigment epithelium-derived factor(PEDF) to cultured human iris pigment epithelial(IPE) cells in vitro to obtain the transgenic cells with high expression of PEDF. Methods Recombinant AAV vector carrying PEDF gene(AAV-PEDF) was constructed,and human IPE cells were cultured in vitro and infected with condensed virus.The expression of green fluorescent protein in cells was observed under fluorescence microscopy,the transfection efficiency was detected by flow cytometry,and the expression of PEDF in IPE cells was detected by RT-PCR and Western blotting. Results The proliferation of IPE cells was normal after infection with AAV-PEDF.The expression of PEDF was observed under fluorescence microscopy and detected by RT-PCR and Western blotting.When the multiplicity of infection(MOI) was 20,the transduction efficiency was 64.22%. Conclusion AAV-PEDF can effectively transfect IPE cells cultured in vitro,and the transgenic cells have a high expression of PEDF.

关 键 词：腺相关病毒 色素上皮衍生因子 虹膜色素上皮细胞 转染 

分 类 号：R773[医药卫生—眼科]