AAV载体介导的视网膜基因转移实验研究  

Experimental study on adeno-associated virus -mediated genetransfer into the rabbit retina

作　　者：单清[1] 张杰[1] 任华[1] 王登龙[1] 伊洪涛[1] 吴小兵[2] 钱焕文[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850 [2]病毒基因工程国家重点实验室,北京100052

出　　处：《眼科新进展》2001年第4期225-227,共3页Recent Advances in Ophthalmology

基　　金：国家 8 63重大项目资助 (编号 :Z2 0 -0 5 -0 2 )

摘　　要：目的　以绿色荧光蛋白 (green fluorescent protein,GFP)作为标记基因 ,观察腺相关病毒 (AAV )载体介导视网膜基因转移作用 ,为视网膜疾病的基因治疗打下基础。方法　制备重组腺相关病毒 r AAV- gfp;将纯化的 r AAV- gfp病毒 10 μL (10× 10 1 2 TU· L- 1 )分别注射于青紫兰灰兔及日本大耳白兔视网膜下 ,于一定时间内取眼球或用眼底照相机进行观察。结果　注射 r AAV- gfp后未见兔眼术后细菌感染及明显免疫反应 ,在荧光显微镜下观察 GFP表达情况 ,3周时可见明显的绿色荧光分布于 RPE细胞层 ,5、6周荧光强度比3周增强 ,荧光范围也有所扩大。直至 10周荧光范围进一步扩大 ,强度更强。眼底照相机观察显示 ,最早 4周观察到眼底 GFP表达 ,至 7个月 (观察仍在继续 )仍然维持在高水平。结论　视网膜下注射 r AAV- gfp病毒可在 RPE细胞内稳定表达报告基因 gfp;AAV载体可望用于视网膜疾病的基因治疗。Objective To evaluate recombinant adeno associated virus (AAV) as an in vivo gene transfer vector for the retina and to explore the possibility of monitoring the expression of green fluorescent protein (GFP) using a flat mounting method and a noninvasive method. Method rAAV gfp were prepared and purified into 10×10 12 TU·L -1 . Chinchilla rabbits and Japanese white rabbits were injected subretinally with 10μL rAAV gfp. Four Chinchilla rabbits were killed severally at 3, 5, 6 and 10 weeks and the eyes were enucleated. The eyecups were cut peripherally in four or five places and flat mounted on a glass slide for fluorescence microscope observation. Three Japanese white rabbits were injected with rAAV gfp for longitudinal studied and the fluorescence was monitored by once a week retinal fluorescence photography. Result The signal was detected after 3 weeks. Gradual fluorescence increase in the number of transduced cells occurred from 3 weeks to 10 weeks. The fluorescence area was larger and the intensity was stronger with time changing. The GFP signal was detected in all the animals as early as 4 weeks. A gradual increase in the number of transduced cells occurred throughout the experimental period of 7 months. Conclusion The subretinal injection has good transduction effect. It is possible to make stable expression of rAAV gfp transduced in the retina. As a marker gene, gfp is suitable for retina AAV transduction.

关 键 词：视网膜 基因转移 腺相关病毒载体 绿色荧光蛋白 AAV GFP 

分 类 号：R774.1[医药卫生—眼科]