内耳疾病基因治疗方法的研究

目的:探讨一种高效、安全和简便的内耳疾病基因转移和导入方法。方法:将30只健康豚鼠随机分为A、B、C 3组,每组10只。用Ad—GFP报告基因分别由圆窗、耳蜗底回和脑脊液三种途径导入30只豚鼠内耳,在术前和术后测定听性脑干反应(ABR)阈值和畸变产物耳声发射(DPOAE)振幅改变。再行耳蜗铺片,观察AdGFP在内耳组织中的转染表达和耳蜗毛细胞的形态结构。结果:三种途径导入Ad—GFP在耳蜗螺旋神经节、血管纹和Corti器上均有转染和表达。在术后3 d的表达产物最高,7 d后减弱,10 d后更弱。圆窗和耳蜗底回钻孔导入手术可引起蜗内出血,凝血块淤积在耳蜗顶回的中阶,造成顶回毛细胞损害。而脑脊液给药途径无此现象。ABR阈值在各组动物手术前后无明显改变,而圆窗和耳蜗底回给药途径造成DPOAE幅度明显下降,脑脊液给药组无DPOAE改变。结论:应用腺病毒载体进行基因转移通过圆窗和耳蜗底回钻孔途径可引起蜗内出血,影响DPOAE幅度,而应用脑脊液给药途径则相对安全和方便。

Methods of gene therapy for inner ear diseases

Objective:To study an effective, safety and convenient method for gene transfer and introduction to inner ear. Method: Ad GFP were transferred into inner ears of 30 guinea pigs through round window, basal turn of cochlea and cerebrospinal fluid respectively. Auditory brainstem response( ABR)and otoacoustic emission of distortion products( DPOAE) were assayed to these animals preoperatively and postoperatively, then transfection and expression of Ad-GFP in inner ears and structure of cochlear hair cells were observed. Result: Transfection and expression of Ad-GFP were found in spiral ganglion of cochlea, stria vascularis and Cortisorgan through all of the three methods. The expression products were in the highest level at the third day after operation. But the operation through round window and basal turn of cochlea could lead to bleeding and coagulum deposited in parietalgy-rus, which could result in hair cells injury. Changes of ABR threshold of these animals were not significant. But amplitude of DPOAE decreased significantly in animals through round window and basal turn of cochlea pathways. There was no hair cells injury and changes of DPOAE through cerebrospinal fluid pathway. Conclusion: The study suggested the operations through round window and basal turn of cochlea could lead to bleeding in cochleas, which resulted in change of DPOAE amplitude. It is a safe and effective method to transfer through cerebrospinal fluid.