腺病毒介导神经生长因子对遗传性视网膜变性RCS大鼠视细胞的营救

Adenovirus mediated nerve growth factor rescues photoreceptor in RCS rats

目的 :探讨先天性视网膜变性病因以及对其进行基因治疗的可行性。方法 :TUNEL法对RCS大鼠视细胞的丧失进行研究 ;构建分泌表达型重组腺病毒穿梭质粒 pAdE1CMV NGF ,经与 pJM17在 2 93细胞中同源重组 ,获得复制缺陷型重组腺病毒AdE1CMV NGF。琼脂糖覆盖法测定滴度。AdE1CMV NGF和AdE1CMV LacZ转导培养视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞。将AdE1CMV LacZ转导组进行x gal染色估计转导效率 ,取AdE1CMV NGF转导组RPE细胞及其条件培养液 ,用RT PCR和WesternBlot进行表达检测 ,用条件培养液刺激PC12细胞 ,证实表达活性。直接进行RCS大鼠视网膜下腔转导 ,一眼注射AdE1CMV NGF ,则另一眼注射AdE1CMV LacZ ,左右眼随机进行。光镜下观察视细胞存活情况。结果 :TUNEL法显示 2 5、35、45、5 5及 6 5dRCS大鼠视网膜视细胞层存在广泛凋亡 ;AdE1CMV NGF的滴度可达 5× 10 10 pfu/ml;4.6× 10 6pfu/ml滴度AdE1CMV LacZ可使RPE细胞发生 10 0 %转导 ;PC12细胞受AdE1CMV NGF转导RPE细胞条件培养液刺激长出突触 ;AdE1CMV NGF视网膜下腔注射使RCS鼠视细胞存活时间明显延长 ,达 15d以上。结论 :视网膜变性模型RCS大鼠视细胞的丧失是以凋亡方式进行 。

Objective: To explore the cause of retinal degeneration and feasibility of gene therapy. Methods: TUNEL assay was used to analyze the loss of photoreceptor in RCS. Shuttle plasmid pAdE1CMV NGF, containing a secretable form of nerve growth factor (NGF), was constructed, and then cotransfected with pJM17 into 293 cells using FuGENE 6 to produce the virion containing the cDNA of interest, AdE1CMV NGF. The viral supernatant was concentrated by ultracentrifugation over cesium chloride, and viral titer was determined by counting plaques using an agarose overlay method. Retinal pigment epithelium (RPE) cells were transduced by AdE1CMV NGF and AdE1CMV LacZ. The amount of released NGF was examined by RT PCR and Western blot. The bioactive evaluation was performed by inducing a marked outgrowth of neurite processes from PC 12 cells. The AdE1CMV NGF and AdE1CMV LacZ were transduced in vivo into subretinal spaces of RCS rat eyes with microsurgery. Results were observed microscopically. Results: TUNEL showed the extensive apoptosis of photoreceptor in 25 , 35 , 45 , 55 and 65 day RCS rat retinae. Subretinal transduction of AdE1CMV NGF delayed photoreceptor cell loss for at least 10 15 days as compared with that transduced by AdE1CMV LacZ, in which only a small number of photoreceptor survived. Conclusion: The loss of photoreceptor of RCS rat retinae is an spoptosis phenomenon; Adenovirusmediated NGF may enhance survival of photoreceptor population of RCS rat retinae.