在体电穿孔辅助基因转染视网膜色素上皮细胞层和光感受器细胞层的实验研究  

Gene transfection into retinal pigment epithelial cells and photoreceptors using in vivo electroporation

作　　者：张萌[1] 莫晓芬[1] 郭文毅[1] 吴继红[1] 方媛[1] 张圣海[1] 李希[2] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院眼科,上海200031 [2]复旦大学上海医学院

出　　处：《中华眼底病杂志》2010年第1期61-65,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金（30672281）;教育部“新世纪优秀人才支持计划”（NCET050370）;上海市青年科技启明星计划（06QA14011）;复旦大学脑科学研究院开放课题;2007年度复旦大学上海医学院基础临床交叉研究基金（JC07-22）;医学神经生物学国家重点实验室开放基金课题（09-07）;上海市青年科技启明星跟踪计划（10QH1400400）

摘　　要：目的探讨在体电穿孔辅助基因转染视网膜色素上皮（RPE）细胞层和光感受器（PR）细胞层的可行性。方法147只健康雄性Spragne—Dawley（SD）大鼠根据电穿孑L刺激模式电压值分为5、10、15、20、25、30、35V组，右眼视网膜下腔注射增强型绿色荧光蛋白（EGFP）真核表达质粒pEGFP—N1为实验眼，左眼注射TE Buffer为对照眼。各组根据不同转染方向再分为RPE和PR两个亚组。各组除电压不同外，其余参数均为脉宽99ms，脉冲间期0．5s，连续5个脉冲。将带有负电荷的质粒在电场作用下，拟转染到RPE细胞层，反向置电极，拟转染到PR细胞层。转染后7d取各组视网膜铺片，荧光显微镜下观察EGFP表达强弱、范围及荧光细胞计数分析；采用蛋白质免疫印迹法（Western blot）、实时逆转录聚合酶链反应（RT—PCR）半定量观察EGFP蛋白和mRNA的表达。结果转染后7d，荧光显微镜观察发现，各组RPE亚组对照眼RPE细胞层内均未见特异性荧光表达，实验眼可见绿色荧光表达；各组RPE亚组对照眼视网膜铺片均未见荧光表达，实验眼视网膜铺片均可见转染了EGFP的RPE细胞。Western blot检测显示，随电压增加，EGFP蛋白与β-肌动蛋白条带灰度相对比值呈上升趋势。RTPCR检测显示，各组均产生阳性扩增条带，随电压增高，EGFP mRNA与GADPH mRNA扩增条带灰度相对比值逐渐增高。结论在体电穿孔方法可以有效辅助基因转染大鼠RPE细胞层。Objective To investigate the feasibility of gene transfection into retinal pigment epithelial （RPE） cells and photoreceptors （PRs） in vivo electroporation. Methods A total of 147 Sprague-Dawley （SD） rats were divided into 5, 10, 15, 20, 25, 30 and 35 V group according to different voltage. The right eyes of rats underwent the injection of eukaryotic expressive plasmid of enhanced green fluorescent protein （EGFP） pEGFP-N1 into subretinal space as experimental eyes; the left eyes were injected with TE buffer as control eyes. Each group was divided into RPE and RP subgroups according to different transfection direction. There were same parameters of 99 ms pulse width, 0.5 s pulse interval and 5 consecutive pulses except different voltage in groups. With a negative charge in the electric field was transfected into RPE cell layer, reverse electrode set to be transfected into PR cell layer. Retina mounts were made on seven days after transfection and the fluorescence of EGFP was photographed by fluorescent microscope. The expression of EGFP mRNA and protein were detected by reverse transcription polymerase chain reaction technique （RT-PCR） and Western blot. Results On seven days after transfection, in RPE subgroups, there were no specific fluorescence expressions in RPE cell layer and retina mounts of control eyes, while there were fluorescence expressions in experimental eyes. Western blot showed that the gray-scale ratio of EGFP protein and β-actin protein bands rose with the increased voltage. RT PCR showed that each group produced positive amplification bands, and the relative ratio of gray level of EGFP mRNA and GADPH mRNA amplified bands gradually increased with the increased voltage. Conclusion Electroporation is an effective method for gene delivery into RPE cells in vivo.

关 键 词：色素上皮 眼 视网膜炎 色素性/治疗 基因疗法 电穿孔 光感受器 动物实验 

分 类 号：R774.1[医药卫生—眼科]