腺相关病毒转导EC-SOD对耳蜗药物性损害的保护作用  

Gene Therapy with Adeno-Associated Virus Vector-Mediated Extracellular Superoxide Dismutase Attenuates Kanamycin-induced Cochlear Damage

作　　者：刘玉和[1] 柯肖枚[1] 钟贞[1] 

机构地区：[1]北京大学第一医院耳鼻咽喉头颈外科,北京100034

出　　处：《听力学及言语疾病杂志》2007年第3期214-218,I0003,共6页Journal of Audiology and Speech Pathology

摘　　要：目的探讨腺相关病毒(adeno-associated virus,AAV)作为载体,介导分泌型超氧化物歧化酶(extracel-lular superoxide dismutase,EC-SOD)基因,转染耳蜗细胞后对卡那霉素致大鼠耳蜗损害的保护作用。方法构建AAV8血清型载体,携载绿荧光蛋白基因(enhanced green fluorescent protein,EGFP)或EC-SOD基因,体外感染细胞,蛋白电泳检测EC-SOD的表达;将AAV8-EGFP和AAV8-EC-SOD分别微注射到卡那霉素耳聋模型大鼠耳蜗内,检测听性脑干反应阈值变化,生化法检测耳蜗外淋巴液EC-SOD酶活性,免疫组织化学检测EC-SOD蛋白在耳蜗细胞内的表达,耳蜗毛细胞和螺旋神经节细胞染色检测细胞存活的数量。结果AAV8转导的绿荧光蛋白可在耳蜗内毛细胞、螺旋韧带细胞、内沟细胞等多种细胞内表达。体外蛋白电泳检测EC-SOD在细胞培养液中存在单体、二聚体、四聚体等;在AAV8-EC-SOD组注射载体的耳蜗外淋巴液中EC-SOD酶活性明显高于AAV8-EGFP组,转导的EC-SOD在耳蜗内的表达分布与绿荧光蛋白的表达相似。在卡那霉素耳聋模型鼠中,AAV8-EC-SOD组的ABR反应阈值变化比AAV8-EGFP组明显小,AAV8-EC-SOD组的耳蜗毛细胞和螺旋神经节细胞比AAV8-EGFP组的存活多,差异有显著统计学意义。结论AAV8型载体可转染耳蜗内多种细胞,AAV8型转导的EC-SOD可以拮抗氨基糖甙类抗生素对耳蜗的损害。Objective To evaluate the otoprotective efficacy of adeno - associated virus serotype 8 （AAV8） as vector mediated extracellular superoxide dismutase （ EC - SOD） gene on kanamycin - induced ototoxicity for cochlear gene therapy. Methods AAV8 vectors harboring the enhanced green fluorescent protein （EGFP） or the rat EC - SOD expression cassette were constructed and prepared. EC - SOD expression in vitro was checked with Western blotting. AAV8 vector was microinjected into the cochlea. The expression patterns of AAV - mediated EGFP and EC - SOD were determined with fluorescence microscope and histochemistry. The cochlear function was analyzed with auditory brain stem responses （ABR）. The cochlear ganglion cells and hair cells were checked for evaluation. Results AAV8 vector had successfully targeted distinct cell types within the cochlea. The monomer, dimer and tetramer of EC - SOD were found in the cell medium inoculated with AAV8 - EC - SOD. EC - SOD activity level in the cochleae inoculated with AAV8- EC- SOD was higher than that in the controls, and EC- SOD expression pattern in the cochlea was similar to AAV8 - mediated EGFP expression. The ABR monitoring revealed that the inoculated cochlea with AAV8 - EC - SOD vector showed better thresholds after exposure to the ototoxins, as compared to the control cochlea. Fewer missing hair cells and spiral ganglion cells were found in the inoculated cochleae, as compared with the controls. Conclusion The results demonstrated the feasibihty of gene therapy with AAV8 as vector for cochlear apphcation, and AAV8 - mediated EC - SOD overexpression preserved the cochlear function from the kanamycin- induced ototoxicity, suggested that AAV8 - mediated expression of EC - SOD may be developed as a valuable prevention against kanamycin - induced damage. The results also supported the hypothesis that oxidant stress plays a significant role in kanamycininduced ototoxicity.

关 键 词：腺相关病毒 耳聋 药物性 超氧化物歧化酶 基因转染/基因治疗 

分 类 号：R764.5[医药卫生—耳鼻咽喉科]