CAJ | 학술논문

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)大鼠根据电穿孔刺激模式电压值分为5、10、15、20、25、30、35 V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99 ms,脉冲间期0.5 s,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7 d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7 d,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.RT-PCR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.
목적 탐토재체전천공보조기인전염시망막색소상피(RPE)세포층화광감수기(PR)세포층적가행성.방법 147지건강웅성Spragne-Dawley(SD)대서근거전천공자격모식전압치분위5、10、15、20、25、30、35 V조,우안시망막하강주사증강형록색형광단백(EGFP)진핵표체질립pEGFP-N1위실험안,좌안주사TE Buffer위대조안.각조근거불동전염방향재분위RPE화PR량개아조.각조제전압불동외,기여삼수균위맥관99 ms,맥충간기0.5 s,련속5개맥충.장대유부전하적질립재전장작용하,의전염도RPE세포층,반향치전겁,의전염도PR세포층.전염후7 d취각조시망막포편,형광현미경하관찰EGFP표체강약、범위급형광세포계수분석;채용단백질면역인적법(Western blot)、실시역전록취합매련반응(RT-PCR)반정량관찰EGFP단백화mRNA적표체.결과 전염후7 d,형광현미경관찰발현,각조RPE아조대조안RPE세포층내균미견특이성형광표체,실험안가견록색형광표체;각조RPE아조대조안시망막포편균미견형광표체,실험안시망막포편균가견전염료EGFP적RPE세포.Western blot검측현시,수전압증가,EGFP단백여β-기동단백조대회도상대비치정상승추세.RT-PCR검측현시,각조균산생양성확증조대,수전압증고,EGFP mRNA여GADPH mRNA확증조대회도상대비치축점증고.결론 재체전천공방법가이유효보조기인전염대서RPE세포층.
Objective To investigate the feasibility of gene transfection into retinal pigment epithelial (RPE)cells and photoreceptors(PRs)in vivo electroporation.Methods A totel 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.