9型腺相关病毒经圆窗膜转导小鼠耳蜗两种方式的对比研究  

Comparison of Two Approaches in Transfection of Adeno-associated Virus Type 9 into Cochlea through Round Window Membrane in Mice

作　　者：齐景翠 祖勉 陈妮姗 郭维维[2] 伊海金[3] 杨仕明[2] 余力生[1] QI Jingcui;ZU Mian;CHEN Nishan;GUO Weiwei;YIN Haijin;YANG Shiming;YU Lisheng(Department of Otolaryngology,Peking University People’s Hospital,Beijing,100044,China;Department of Otolaryngology Head and Neck Surgery,Chinese PLA General Hospital,Chinese PLA General Hospital Institute of Otolaryngology,Key Laboratory of Hearing Impairment Science(Chinese PLA Medical School),Ministry of Education Beijing Key Laboratory of Hearing Impairment Prevention and Treatment,Laboratory of Military Acoustic Trauma Protection;Department of Otolaryngology Head&Neck Surgery,Beijing Tsinghua Changgung Hospital)

机构地区：[1]北京大学人民医院耳鼻咽喉科,100044 [2]中国人民解放军总医院耳鼻咽喉头颈外科,解放军总医院耳鼻咽喉研究所,聋病教育部重点实验室,聋病防治北京市重点实验室,军事声损伤防护实验室 [3]清华大学附属北京清华长庚医院耳鼻咽喉头颈外科

出　　处：《中华耳科学杂志》2018年第4期469-473,共5页Chinese Journal of Otology

基　　金：首都卫生发展科研专项重点攻关项目(2016-1-5014);国家自然基金面上项目(81670940);军事医学创新工程和青年培育项目(16QNP133);全军后勤重点项目(BWS14J045)Militarynoisegrant(BWS14J045);北京市属医院科研培育计划项目-PX2017046资助。

摘　　要：目的通过圆窗膜显微注射和完整圆窗膜途径转导小鼠内耳携带GFP的9型腺相关病毒(adeno-asso-ciated virus,AAV),比较两种方式的可行性、转染效率以及对听力的影响。方法 18只C57BL/6小鼠,随机分为圆窗膜显微注射组和完整圆窗膜途径组,术前均行听性脑干反应(auditory brain response, ABR)测听,术后两周行ABR测听后取双侧耳蜗基底膜铺片及冰冻切片观察GFP表达情况。结果两种转导方法对小鼠听力均无显著影响,圆窗膜显微注射组转染耳蜗内毛细胞GFP可见表达,转染效率由底转到顶转逐渐降低,无毛细胞损伤,经完整圆窗膜途径转染耳蜗未见GFP表达。结论 9型AAV难以通过完整圆窗膜,通过显微注射方式能够将目的基因高效转导内耳,对内毛细胞有选择特异性,且对听力影响较小,是一种安全有效的内耳转导方式。Objective To compare the feasibility,transfection efficiency and impact on hearing between trans-round window membrane(RWM)microinjection and intact RWM delivery of GFP into the inner ear via adeno-associated virus(AAV)type 9.Methods Eighteen C57BL/6 mice were randomly chosen to receive trans-RWM microinjection or intact RWM delivery.Auditory brain response(ABR)thresholds were determined before and two weeks after operation.Animals were sacrificed and specimens collected for examination of GFP expression.Results Neither method had deleterious effect on hearing.High levels of GFP expression in inner hair cells were seen only in the microinjection group,with transfection efficiency gradually decreasing from the base to the apex.There was no hair cell damage.Conclusion AAV type 9 is hard to deliver through the intact RWM.Trans-round window membrane microinjection is a safe and efficient delivery approach into the inner ear.AAV type 9 shows high specificity to inner hair cells with little impact on hearing.

关 键 词：9型腺相关病毒 圆窗膜 内耳入路 基因转导 

分 类 号：R764[医药卫生—耳鼻咽喉科]