重组腺病毒Ad-EGFP转染大鼠角膜的实验研究  

Experimental studies of recombination adenovirus Ad-EGEP transfecting rat corneas

作　　者：袁伟[1] 陆晓和[2] 宫玉波[2] 周瑾[2] 

机构地区：[1]济南军区青岛第一疗养院体检科,山东省青岛市266071 [2]南方医科大学珠江医院眼科,广东省广州市510280

出　　处：《眼科新进展》2011年第4期324-326,331,共4页Recent Advances in Ophthalmology

基　　金：广东省科技计划项目资助(编号:2006B36003008)~~

摘　　要：目的评价携带报告基因的重组腺病毒Ad-EGFP经不同转染途径原位转染大鼠角膜的可行性和安全性,探索重组腺病毒载体原位转染大鼠角膜的最佳途径。方法取SD大鼠80只,随机分为A、B、C、D4组,A组、B组:分别于球结膜下注射Ad-EGFP15μL及9g·L-1NaCl注射液15μL;C组、D组:分别前房注射Ad-EGFP15μL及9g·L-1NaCl注射液15μL。各组大鼠分别于注射后1d、5d、7d及14d摘除角膜,行HE染色观察角膜组织有无超微结构改变,共聚焦显微镜观察荧光表达,透射电镜观察有无病毒颗粒存在。结果 4组大鼠于注射当时及整个观察期内角膜组织各层均未见明显超微结构改变,眼前段组织未见明显的组织病理性改变;术后B、D组角膜始终未见绿色荧光的阳性表达,电镜下亦未见病毒颗粒;A、C组注射后1d角膜即可见绿色荧光;5d荧光表达最强,7d时较5d时减弱,14d时更弱。且注射1d后电镜下可见腺病毒颗粒;A组绿色荧光表达位于角膜上皮层及角膜基质层;C组绿色荧光表达仅位于角膜内皮层,角膜上皮层及基质层未见阳性表达。结论携带报告基因的重组腺病毒Ad-EGFP可通过球结膜下注射、前房注射等途径原位转染至大鼠角膜组织,且安全性好;球结膜下注射可使腺病毒携带的报告基因在角膜上皮层及角膜基质层表达;前房注射途径可使腺病毒携带的报告基因在角膜内皮层表达。Objective To investigate the efficacy and safety of in situ transfection of recombinant adenovirus Ad-EGFP with the report gene to rat corneas through different ways and seek for the best method of in situ transfection of recombinant adenovirus vector to rat corneas. Methods About 80 SD rats were divided into 4 groups randomly.Rats in group A and group B were bulbar subconjunctival injected with 15 μL Ad-EGFP and 15 μL 9 g·L-1 sodium chloride,respectively.Rats in group C and group D were injected with 15 μL Ad-EGFP and 15 μL 9 g·L-1 sodium chloride into anterior chamber,resepectively.Corneas were removed at 1 day,3 days,5 days,7 days and 14 days after injection.HE staining was used to observe the microstructural changes of corneal tissues;Confocal microscope was used to observe fluorescence expression;Transmission electron microscope was used to detect there was viral particle or not. Results Obvious ultrastructural changes were not found in each layer of corneal tissues at the time of injection and the whole observation time in all 4 groups.Obvious histopathological changes were not found in anterior segment;After the operation,positive expression of green fluorescence was not found in corneas of group B and group D;Viral particles were not found under electron microscope.Green fluorescence could be found in group A and group C at 1 day after injection.Fluorescence went to peak at 5 days,fluorescence at 7 days was weaker than that at 5 days,and was the weakest at 14 days.Adenoviral particles were found at 1 day after injection under electron microscope;Green fluorescence was found at corneal epithelial layer and stroma in group A.Green fluorescence was only found in corneal endothelial layer,but not found in corneal epithelial layer and stroma in group C. Conclusions Recombinant adenovirus Ad-EGFP with the report gene can in situ transfect to corneal tissues with sub-conjunctival injection and injection into anterior chamber,and it is safe.Expression of report gene in adenovirus can be found in corneal epi

关 键 词：重组腺病毒 绿色荧光蛋白 角膜 大鼠 

分 类 号：R772.2[医药卫生—眼科]