摘要
目的 研究重组腺相关病毒 (rAAV)载体介导的绿色荧光蛋白基因 (GFP)对体外培养虹膜色素上皮 (IPE)细胞的转染和表达 ,为rAAV携带目的基因治疗视网膜色素变性提供理论依据。方法 酶辅助的显微分离方法体外培养IPE细胞并鉴定。按转染倍数 (MOI)为 10 3 、10 4、10 5、10 6转染已培养 3d的传二代IPE细胞 ,转染后的第 1、3、5、7、9d ,在倒置荧光显微镜下观察IPE中GFP表达阳性的情况 ,记录 10 0个IPE细胞中GFP阳性所占的百分比。当GFP表达稳定后 ,共聚焦显微镜照相 ,流式细胞仪检测rAAV GFP对IPE的转染效率。结果 rAAV GFP在细胞中的表达呈绿色 ,弥漫于整个胞浆。MOI =10 3 时 ,转染后 48h ,GFP开始表达 ,MOI =10 4、10 5、10 6时 ,转染后 2 4h时便可看到GFP表达阳性的细胞 ,细胞的起始表达强度不一 ,随MOI值的增高而增强 ,同时GFP的表达强度随着时间延长而逐渐增强 ,转染后第 7～ 9d达到高峰并维持 ,此时检测rAAV GFP对IPE的转染效率 ,MOI =10 3 时是 2 6 7% ,MOI =10 4时是 5 3 6% ,MOI =10 5时是 60 2 % ,MOI =10 6时是 63 7%。结论 rAAV GFP对体外培养的IPE细胞转染效率可达 60 %以上 。
ObjectiveTo evaluate the efficiency of recombinant adeno associated virus(rAAV) mediated transduction of green fluorescent protein(GFP) gene to iris pigment epithelial(IPE) cells in vitro.MethodsIPE cells were cultured and identified by immunocytochemical staining.According to various multiple of infection(MOI=10 3 ?10 4 ?10 5 ?10 6 ), rAAV GFP were added to the 2th generation of IPE cells cultured for three days. After IPE cells were transducted for 1,3,5,7,9 days,the inverted microscope observation of fluorescence was performed to assess GFP expression and the percentage of positive GFP in random 100 IPE cells was calculated.The transudation efficiency of rAAV GFP to IPE was evaluated by Flow Cytometer and Confocal microscope.ResultsThe GFP expression showed green fluorescence in the cytoplasm, GFP expression in IPE cells could be detected in 24 h (MOI=10 4 ,10 5 ,10 6 ) and 48 h(MOI=10 3 ) after transduction.Expression increased with the increase of MOI and reached fastigium in 7～9 days.At that time,the transduction efficiency was 26 7%(MOI=10 3 ),53 6%(MOI=10 4 ),60 2%(MOI=10 5 ),63 7% (MOI=10 6 ).ConclusionThe efficiency of rAAV mediated transduction of GFP gene to IPE cells is above 60%.It provides the possibility of combining rAAV mediated gene therapy with IPE transplantation to treat retinitis pigmentosia(RP).
出处
《眼科研究》
CSCD
北大核心
2003年第2期160-162,共3页
Chinese Ophthalmic Research
关键词
腺相关病毒
绿色荧光蛋白基因
虹膜色素上皮细胞
体外
转染
adeno associated virus green fluorescent protein iris pigment epithelial cells in vitro transduction
