新霉素对大鼠前庭的损伤及复制缺陷型腺病毒前庭导入的研究

Neomycin Induced Damage to Rat Vestibules and Gene Transfer into Vestibule Using Repliation Defective Adenovirus

徐金操;胡吟燕;徐延军;黄德亮;杨仕明;

1:解放军总医院耳鼻咽喉-头颈外科解放军耳鼻咽喉科研究所耳神经生物中心

摘要

目的研究新霉素对大鼠前庭毛细胞的毒性作用并探索前庭基因导入的方法和途径,为药物性前庭功能损伤动物模型的制备和基因治疗的相关研究提供参考。方法将25只成年Wistar大鼠分为正常对照组、新霉素鼓阶给药组、新霉素前庭阶给药组、腺病毒(缺失E1、E3基因且构建有绿色荧光蛋白报告基因的复制缺陷型腺病毒,Ad-EGFP)鼓阶导入组、腺病毒(Ad-EGFP)前庭阶导入组,每组各5只动物。新霉素组大鼠在右耳通过耳蜗底回鼓阶(鼓阶组)或前庭阶打孔(前庭组)的方法在耳蜗内注入0.1%新霉素5μl,Ad-EGFP组则以同法导入物理滴度为2.1×1011vp/ml的Ad-EGFP 5μl,正常对照组大鼠不做任何处理。处理3天后对动物进行颈髓硬膜外短声诱发电位(click-evoked potentials on the surface of the cervical dura mater,CDM-CEP)检查和游泳试验,评价前庭功能,然后将动物处死,进行组织学和形态学观察。结果3天后新霉素组大鼠的前庭毛细胞即出现严重破坏,并出现严重的前庭功能损伤。正常对照组、新霉素鼓阶给药组、新霉素前庭阶给药组、Ad-EGFP鼓阶导入组、Ad-EGFP前庭阶导入组的游泳时间分别为4.0±0.71、10.2±1.64、9.8±1.48、4.8±0.84、5.0±0.71 s;正常对照组CDM-CEP的阈值为85±3.54 dB SPL,潜伏期为6.59±0.41 s;新霉素组120 dB SPL未引出CDM-CEP;Ad-EGFP鼓阶导入组CDM-CEP阈值为91±5.48 dB SPL,潜伏期为6.76±0.26 s,Ad-EGFP新霉素前庭阶导入组CDM-CEP的阈值为89±6.52 dB SPL,潜伏期为6.78±0.26 s。3天后,Ad-EGFP前庭阶导入组的前庭终末器官均出现Ad-EGFP的转染;而Ad-EGFP鼓阶导入组未见前庭终末器官的表达。结论新霉素对前庭毛细胞具有极强的破坏作用,新霉素耳蜗内注射的方法可以制备理想的前庭功能障碍动物模型,耳蜗底回前庭阶打孔可以作为基因导入前庭的有效途径。

关键词

新霉素;前庭;毛细胞;基因导入

基金项目(Foundation):

国家自然科学基金面上项目(3057101730000189);; 海外青年合作基金(30628030);; 北京自然科学基金(7042061);; 国家863计划(2007AA02Z150);; 国家自然科学基金重点项目(30730040)资助

作者

徐金操;胡吟燕;徐延军;黄德亮;杨仕明;

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徐金操胡吟燕徐延军黄德亮杨仕明