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目的:带绿色荧光蛋白(GFP)基因的重组腺病毒,为利用重组腺病毒介导外源基因转导内耳提供依据。方法:通过细菌内同源重组方法构建携带有报告基因的重组腺病毒(Ad-GFP),将重组腺病毒经耳蜗底回途径导入豚鼠耳蜗鼓阶外淋巴后,观察手术后不同时间GFP基因在豚鼠耳蜗内的表达、分布情况;检测手术前后听觉脑干诱发电位,观察手术和病毒对豚鼠听觉功能的影响。结果:构建的重组腺病毒经酶切电泳鉴定正确;豚鼠耳蜗底回途径导入腺病毒24 h后即有报告基因表达,3 d后最高,表达时间可持续2周以上;报告基因表达部位主要分布在血管纹、鼓阶外淋巴面的间皮细胞和耳蜗Corti器等部位;听觉脑干诱发电位(ABR)在各组动物手术前后无明显改变。结论:成功构建了携带GFP基因的重组腺病毒,通过该方法构建的腺病毒可以介导报告基因在豚鼠耳蜗中表达,并且对豚鼠听觉功能无明显影响,为内耳基因治疗提供了理论依据。
Objective: Recombinant adenovirus carrying green fluorescent protein (GFP) gene provides the basis for transducing the inner ear with recombinant adenovirus. Methods: Recombinant adenovirus (Ad-GFP) carrying the reporter gene was constructed by homologous recombination in bacteria. The recombinant adenovirus was transduced into the extra-amphical lymph of the cochlea of the guinea pig through the cochlear gyrus, and the expression of GFP gene at different time The expression and distribution of guinea pig cochlea were observed. The auditory brainstem response was measured before and after surgery to observe the effects of surgery and virus on the auditory function of guinea pigs. Results: The constructed recombinant adenovirus was identified by enzyme-digestion electrophoresis. The expression of the reporter gene was induced in the bottom of the cochlea of the guinea pig by 24 h after transduction. The expression of the reporter gene was highest after 3 days and the expression time was longer than 2 weeks. There was no significant change in auditory brainstem response (ABR) before and after auditory brain stem evoked potentials in vascular pattern, mesothelial cells on the outer surface of the tympanum, and Corti apparatus of the cochlea. CONCLUSION: The recombinant adenovirus carrying GFP gene was successfully constructed. The adenovirus constructed by this method can mediate the expression of the reporter gene in guinea pig cochlea, and has no obvious effect on the auditory function of guinea pigs. It provides a theoretical basis for the gene therapy of the inner ear.