不同病毒载体感染内耳组织的比较研究  

Comparison of gene transfer into the cochlea using adeno-associated virus versus adenovirus vectors

作　　者：杨阳[1] 孔维佳[1] 胡钰娟[1] 钟毅[1] 赵学艳[1] 彭炜[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院耳鼻咽喉-头颈外科,武汉430022

出　　处：《中华医学杂志》2009年第19期1351-1355,共5页National Medical Journal of China

基　　金：国家自然科学基金（30730094）;国家科技部“十一五”科技攻关项目基金（2007BM18813）;国家杰出青年基金（39925035）

摘　　要：目的通过血清5型重组腺病毒和血清2型重组腺相关病毒感染大鼠内耳组织的比较研究，筛选出更适合内耳基因治疗的病毒载体。方法一定体积的血清5型重组腺病毒液和血清2型重组腺相关病毒液经圆窗膜注入鼓阶外淋巴液，通过荧光共聚焦显微镜、免疫组化、Western免疫印迹及听性脑干反应等方法对两种载体所携带报告基因的表达部位、表达时程以及两种病毒本身对内耳组织细胞生活状态的影响加以比较。结果血清5型重组腺病毒和血清2型重组腺相关病毒所携带的增强型绿色荧光蛋白报告基因均可在前庭膜、血管纹、基底膜、盖膜、螺旋神经节区及转染对侧耳表达。rAAV2携带的EGFP蛋白第14天表达开始明显增多，第60天仍可检测到高表达。rAd5携带的EGFP蛋自在耳蜗组织内的表达高峰维持在第1～21天，在30天时表达明显减弱。结论腺病毒的优势在于其基因表达的迅速和高效，而腺相关病毒载体以其长的表达时程，低组织细胞毒性在内耳基因转染中表现出独特的优势。Objective To determine which of the two,recombinant adeno- associated viral vector 2 （rAAV2） and recombinant adenovirus vector 5 （ rAd5 ）, is more suitable for gene transfer in rodent cochlea. Methods The rAAV2-EGFP and rAd5-EGFP partiels were injected into the perilymph through round window membrane. The target tissue accessibility, time course of expression, tissue toxicity of gene transfer and effects on heating were evaluated. Results The expression of EGFP was detected in spiral ligament, strial vascukarises, Reissner membrane, basilar membrane, spiral ganglion, and eontralateral cochlea. EGFP expression in the rAAV2 lasted over 60 days, with peak expression between days 14 and 60. EGFP in the rAd5 was detected within 24 h of transfection, and peak expression was observed between days 1 to 21. EGFP activity decreased sharply on day 30 after transfection with rAd5, while high EGFP expression was observed 60 days after transfection with rAAV2. Conclusion AAV has significant advantages for long- term transgene expression and no ototoxicity in the cochlea compared to adenovirus vectors.

关 键 词：腺病毒 腺相关病毒 增强型绿色荧光蛋白 内耳 

分 类 号：R686[医药卫生—骨科学]