重组腺病毒构建及介导报告基因在豚鼠耳蜗中的表达

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。

Construction of recombinant adenovirus and mediated reported gene expression in the guinea pig cochlea

Objective:To purify P0 protein from guinea pig's inner ear by preparative SDS-PAGE and study the possible role it may play in the etiology of autoimmune inner ear disease.Method:A mixture of membraneous proteins of inner ear was separeted by preparative SDS-PAGE.The corresponding band at 30kd was cut and electrally eluted.The protein collected was identified by analytical SDS-PAGE and Western blot assay.A group of 20 guinea pigs were immunized with P0 protein emulsified in complete Freund's adjuvant,another 10 guinea pigs were immunized with complete Freund's adjuvant only as control.The guinea pigs' hearing thresholds,serum IgG level and morphological changes in the inner ear were investigated.The distribution of P0 protein in the cochlear was detected by immunohistochemical technique.Result:The purity of the protein was demonstrated by a single band at the 30 kD site in SDS-PAGE,which was identified as P0 protein by western blot analysis assay.About 17.5% P0-immunized guinea pigs showed increased hearing thresholds,elevated IgG level(F=6.48,P<0.01),as well as a decreased number of spiral ganglion cells and inflammatory cell infiltration in the cochlear nerve region.The P0 protein is distributed in the cochlear nerve and spiral ganglion only.Conclusion:P0 protein from guinea pig's inner ear can be successfully purified by preparative SDS-PAGE and an animal model of experimental autoimmune inner ear disease induced by P0 protein is successfully established.