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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 ,腺病毒介导神经生长因子可延长其存活时间
Objective: To explore the etiology of congenital retinal degeneration and its feasibility of gene therapy. Methods: TUNEL method was used to study the loss of visual cells in RCS rats. The recombinant adenovirus shuttle plasmid pAdE1CMV NGF was constructed and homologously recombined with pJM17 in 293 cells to obtain recombinant adenovirus AdE1CMV NGF. Agarose assay to determine titer. AdE1CMV NGF and AdE1CMV LacZ transduced retinal pigment epithelium (RPE) cells. The transduction efficiency of AdE1CMV LacZ transduction group was evaluated by xgal staining. The RPE cells and their conditioned medium of AdE1CMV NGF transduction group were detected by RT PCR and Western Blot. The conditioned medium was used to stimulate PC12 cells to confirm the expression activity. Direct RCS rat subretinal transduction, one injection of AdE1CMV NGF, the other injection of AdE1CMV LacZ, left and right eyes were randomized. Light microscopic observation of cell survival. Results: TUNEL assay showed extensive apoptosis in the retinal cell layer of 2 5, 35, 45, 55 and 65 dRCS rats. The titer of AdE1 CMV NGF was 5 × 10 10 pfu / ml and 4.6 × 10 6 pfu / ml AdE1CMV LacZ induced 100% transduction of RPE cells; PC12 cells were stimulated by AdE1CMV NGF-transduced RPE cells conditioned medium to grow synapses; AdE1CMV NGF subretinal injection significantly prolonged the survival time of RCS mice, reaching 15 days the above. Conclusion: Retinal degeneration model RCS rat visual cell loss is based on apoptosis, adenovirus-mediated nerve growth factor can prolong its survival time