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

刘玉和;柯肖枚;钟贞;

1:北京大学第一医院耳鼻咽喉头颈外科

2:北京大学第一医院耳鼻咽喉头颈外科

3:北京大学第一医院耳鼻咽喉头颈外科 北京100034

4:北京100034

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。
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
基金项目(Foundation):
作者
刘玉和;柯肖枚;钟贞;
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