NT-3基因转染对耳蜗传入神经损伤保护作用电镜观察

Ultrastructural evaluation of NT-3 gene transfection on the guinea pig afferent nerve system treated with(gentamicin)

目的探讨阳离子脂质体介导的神经营养素-3(NT-3)基因转染对庆大霉素性耳蜗传入神经损伤超微结构的影响。方法采用阳离子脂质体介导法,将携带外源性NT-3基因的重组真核表达载体增强型绿色荧光蛋白(pIRES2-EGFP)-NT-3注入豚鼠左侧耳蜗,术后3 d开始给予庆大霉素肌注(120 mg/kg),连续注射14 d,分别于停药后1 d、2周进行听性脑干诱发反应(ABR)测听,随即处死动物,并进行耳蜗取材,在透射电镜下观察耳蜗传入神经超微结构改变。结果庆大霉素停药1 d时,与正常对照组相比,空载体及磷酸盐缓冲液(PBS)组ABR阈值显著提高(P<0.01),NT-3治疗组ABR阈值增高虽不如前二者显著,但仍有差异(P<0.05),但以上3组间无显著性差异;当庆大霉素停药2周时,NT3治疗组ABR阈值虽仍有提高,但与停药1 d时相比,无显著性差异(P>0.05),而空载体及PBS组ABR阈值升高显著(P<0.05)。透射电镜下见庆大霉素停药1d时,空载体及PBS组传入性神经末稍已出现肿胀,突触间隙明显增大,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型神经元胞体内偶可见少量髓鞘样结构;在停药2周时,传入神经退行性改变明显加重,出现突触结构消失、空泡化,神经纤维崩解,神经元出现大量空泡化。而NT3基因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2周时,神经退行性改变略加重,但仍显著轻于空载体及PBS组对照组。结论阳离子脂质体介导的NT-3基因转染对豚鼠庆大霉素性耳蜗传入神经性损伤具有一定程度的保护作用。

Objective To study the protection of neurotrophin-3 （NT-3） gene transfection on the guinea pig cochlear afferent nerve system treated with gentamicin. Methods The liposome plasmid complex （plRES2-EGFP （enhanced green fluorescent protein）-NT3） was injected into the guinea pig cochlea, three days later, the guinea pigs were treated with gentamicin （120 mg/kg, 14 d）. The acoustic brainstem evoked response （ABR） was measured at 1 d and 2 w after stopping the genetanmicin. After the cochlear was harvested, changes in the afferent nerve ending, afferent nerve fibers and cell body were evaluated by transmission electron microscopy respectively, Results One day after stopping gentamicin, compared with those of the nornal control group, the thresholds of ABR of the guinea pigs transfected with blank vector and phosphate buffered saline （PBS） decreased evidently（ P 〈0.01 ）, and two weeks after stopping gentamicin, the thresholds decreased much（P 〈0.05, compared with those of NT3 transfection group）. While the ABR thresholds of the group transfected with NT3 dropped at 1 d （P 〈0.05, compared with those of the normal group）, at 2 w, the ABR thresholds dropped slowly （P 〉0.05, compared with those of NT3 transfecton group at 1 d）. One day after stopping gentanmicin, the hair cells and afferent synapses beneath hair cells were swollen firstly, then the nerve fibers and spiral ganglion ceils in the groups treated with blank vector and PBS. At 2 w, the hurt became more seriously, while the changes in group transfected with NT-3 were small. Conclusion Cochlear NT- 3 gene transfection can provide safe and effective protection to the guinea pig cochlear afferent nerve system treated with gentamicin.