腺伴随病毒携带神经营养因子-3对庆大霉素致聋豚鼠耳蜗的保护作用

Protective effect of adeno-associated virus-mediated neurotrophin-3 on the cochlea of guinea pigs with gentamicin-induced hearing loss

目的探讨采用腺伴随病毒(AAV)携带神经营养因子-3(NT-3)对庆大霉素(GM)致聋豚鼠的局部基因治疗及其对耳蜗功能及形态的保护作用。方法选用18只杂色健康豚鼠,用GM按80mg/kg·d连续肌肉注射4d后随机分为3组:AAV-NT-3治疗组(A组)7只,AAV对照组(B组)7只,GM对照组(C组)4只。A、B组动物在埋植微量渗透泵后继续肌肉注射GM10d,C组动物继续注射GM10d。AAV-NT-3及空白AAV载体滴度均为1.1×1010pfu/ml。对A、B两组注射GM前及术后10d、C组注射GM后14d进行听性脑干反应测听(ABR)及畸变产物耳声发射(DPOAE)测定。而后将动物麻醉处死取听泡,行基底膜铺片和毛细胞计数,计算外毛细胞损失率。结果实验组与对照组相比听功能(ABR及DPOAE)及耳蜗毛细胞生存率均有显著性差异(P<0.05)。结论腺伴随病毒介导NT-3转基因治疗可用于保护受氨基甙类药物损害的豚鼠听功能和耳蜗毛细胞;在行局部耳蜗转基因治疗中应严格遵循无菌原则。

Objective To study the protective effect of local gene therapy with adeno-associated virus （AAV）-mediated neurotrophin-3 （NT-3） on the function and morphology of the cochlea of guinea pigs with gentamicin-induced hearing loss. Methods Hearing loss was induced with gentamicin （80 mg·kg^-1·day^-1 injected intramuscularly） in 18 pigmented guinea pigs 4 days prior to gene transfer. The guinea pigs were then divided into groups A, B, and C for AAV-mediated NT-3 gene transfer （n=7）, AAV infection （n=7） or no particular intervention （n=4）, respectively. Mini-Osmotic pump were imolanted in either side of the ears in groups A and B, and the guinea pigs were injected with gentamicin （80 mg·kg^-1·day^-1） intramuscularly since the operation day for 10 consecutive days. In group C, only gentamicin was administrated. Before and 14 days after gentamicin administration, auditory brainstem response audiometry （ABR） and distort-product otoacoustic emissions （DPOAE） were recorded, and the animals sacrificed to observe the morphological changes of the cochlear microscopically. Results Compared with groups B and C, the animals in group A showed better auditory ability （ABR and DPOAE） and significantly highersurviving rate of the outer hair cells （P〈0.05）. Conclusion AAV-mediated NT-3 gene transfer may protect and repair the cochlear hair cells and auditory function damaged by arninoglycoside ototoxicity in guinea pigs, and aseptic procedure id of vital importance in cochlear local gene therapy.