NT-3基因转染对庆大霉素耳毒性的拮抗作用  

Protective Role of NT-3 Gene Transfection on the Guinea Pig Cochlea Treated with Gentamicin

作　　者：邓志宏[1] 王锦玲[1] 邱建华[1] 刘顺利[1] 黄晓峰[2] 黄梅[2] 王成济[3] 杨安钢[3] 

机构地区：[1]第四军医大学西京医院耳鼻咽喉科,西安710032 [2]第四军医大学电镜室 [3]第四军医大学生化教研室

出　　处：《听力学及言语疾病杂志》2006年第4期285-288,T0002,共5页Journal of Audiology and Speech Pathology

摘　　要：目的探讨NT-3基因转染对豚鼠庆大霉素耳中毒的保护作用。方法采用阳离子脂质体介导的方法,将携带外源性基因NT-3的重组质粒pIRES2-EGFP-NT-3与阳离子脂质体复合物10μl注入豚鼠左侧耳蜗(设为Ⅰ组),术后3d开始给予庆大霉素肌肉注射(120mg/kg),连续注射14d,分别于停药后1d、2w进行ABR及DPOAE测试,随即处死动物,耳蜗取材,观察耳蜗毛细胞受损情况,并在透射电镜下观察耳蜗传入神经超微结构改变。实验同时设空载体组(Ⅱ组)及正常对照组(Ⅲ组)。结果庆大霉素停药1d时,与正常对照组相比,空载体组DPOAE幅值显著下降(P<0.05),停药2w时,DPOAE幅值下降更显著,尤其在1、1.4及6kHz处幅值下降明显(P<0.05);NT-3转染组在停药1d时,仅高频DPOAE幅值下降(P<0.05),停药2w时,DPOAE幅值下降减缓(P>0.05);各实验组ABR阈值与DPOAE幅值改变相似;荧光显微镜下见庆大霉素停药1d时,NT-3转染组第一排外毛细胞出现部分缺失,庆大霉素停药2w时,第一排外毛细胞缺失加重,并出现少量第二、三排外毛细胞及个别内毛细胞的缺失;空载体组在庆大霉素停药1d时,即出现第一排外毛细胞严重缺失,少量第二、三排外毛细胞及个别内毛细胞的缺失,停药2w时外毛细胞缺失进一步加重;透射电镜下见庆大霉素停药1d时,空载体对照组Ⅰ型及Ⅱ型神经纤维轴索内微管排列出现紊乱,少量崩解,髓鞘板层结构尚可,Ⅰ、Ⅱ型神经元细胞胞体内偶可见少量髓鞘样结构,停药2w时,传入神经退行性改变明显加重,神经纤维崩解,神经元出现大量空泡化。而NT-3转染组在损伤的早期,传入性神经从末梢到神经元仅出现轻微的改变,在庆大霉素停药2w时,神经退行性改变略加重,但仍显著轻于对照组。结论阳离子脂质体介导的NT-3基因转染可减轻庆大霉素对豚鼠耳蜗外毛细胞、传入神经及螺旋神经节细胞的毒性作用。Objective To study the protective role of NT- 3 gene transfection on the guinea pig cochlea treated with gentamicin. Methods The liposome- plasmid complex （pIRES2 - EGFP- NT- 3） was injected into the guinea pig cochlea（group Ⅰ ）, three days later the guinea pigs were treated with gentamicin （120 mg/kg, 14 d） .ABR and DPOAE were measured at ld and 2w after stopping the genetamicin. After the cochlea were harvested, changes in the hair cells, afferent nerve fibers and cell body were evaluated with FITC- phalloidin staining and transmission electron microscopy respectively. Results At ld after stopping gentamicin, compared with the normal group, the DPOAE amplitudes of the guinea pigs transfected with blank vector were noticeably reduced（P 〈 0.05）, and at 2w more reduction was noted, especially at 1.0,1.4 and 6 kHz（P 〈 0.05）. While only the high frequency, the DPOAE amplitudes of the guinea pigs transfected with NT - 3 decreased at 1 d （ P 〈 0.05）, at 2w the DPOAE responeses were slowly reduced（ P 〉 0.05）. The ABR thresholds were similar to the results of DPOAE. At ld after stopping gentamicin, the hair cells were lost evidently, the nerve fibers and spiral ganglion cells in the groups treated with blank vector became swollen. In two weeks after stopping gentamicin,more losses were abserved. The changes in groups transfected with NT- 3 were insignificant. Conclusion Cochlear NT - 3 gene transfection can provide safe and effective protection to the guinea pig cochlea treated with gentamicin.

关 键 词：NT-3 基因转染 耳蜗 庆大霉素 传入神经 

分 类 号：R764.5[医药卫生—耳鼻咽喉科]