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目的研究携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)腺病毒(Ad-EG-FP)经由小鼠耳后圆窗径路导入内耳的可行性,分析EGFP在耳蜗内的表达特点。方法 19只健康的8～9周龄C57BL/6J雄性小鼠随机分为三组:Ad-EGFP组7只,人工外淋巴液组6只,两组通过耳后切口圆窗径路注射,分别导入Ad-EGFP和人工外淋巴液;空白对照组6只,未予处理。各组均于术前3日和术后7日行听性脑干反应(ABR)检查;术后7日取出耳蜗于荧光显微镜下观察EGFP在内耳的分布并行免疫组化观察EGFP在基底膜的表达。结果 3组动物术前ABR反应阈差异无统计学意义(P>0.05),术后7日,Ad-EGFP组ABR反应阈为62.86±9.94dBSPL,人工外淋巴液组ABR反应阈为60.83±9.70dBSPL,均较术前(37.86±8.59和34.16±8.04dBSPL)及空白对照组(40.83±8.61dBSPL)高(P值均<0.05);空白对照组实验前后ABR反应阈无变化,Ad-EGFP组与人工外淋巴液组术后7天ABR反应阈差异无统计学意义(P值均<0.05)。Ad-EGFP组Ad-EGFP导入后在基底膜上可见EGFP呈广泛表达,人工外淋巴液组和空白对照组基底膜未见荧光表达。结论外源性基因可经内耳圆窗导入并在耳蜗基底膜上广泛表达。
Objective To investigate the feasibility of introducing EGFP adenovirus (Ad-EG-FP) into the inner ear through the mouse ear circular window and to analyze the expression of EGFP in the cochlea. Methods Nine healthy C57BL / 6J male mice aged 8-9 weeks were randomly divided into three groups: 7 in the Ad-EGFP group and 6 in the group of artificial perilymph. The two groups were injected through the posterior ear incision circular window, Ad-EGFP and artificial perilymph; blank control group 6, untreated. All patients underwent auditory brainstem response (ABR) 3 days before surgery and 7 days after surgery. The cochlea were removed on the 7th day after surgery to observe the distribution of EGFP in the inner ear. Immunohistochemistry was performed to observe the expression of EGFP in the basilar membrane expression. Results There was no significant difference in preoperative ABR threshold between the three groups (P> 0.05). On the 7th postoperative day, ABR threshold was 62.86 ± 9.94 dBSPL in Ad-EGFP group and 60.83 ± 9.70 in artificial perilymph group dBSPL were significantly higher than those in the control group (37.86 ± 8.59 and 34.16 ± 8.04dBSPL) and the blank control group (40.83 ± 8.61dBSPL) (P <0.05) There was no significant difference in ABR threshold between the two groups (P <0.05). Ad-EGFP Ad-EGFP after the introduction of EGFP was found in the basal membrane was widely expressed, artificial perilymph and blank control group, no fluorescence expression of the basement membrane. Conclusion The exogenous gene can be introduced into the inner ear round window and widely expressed on the basement membrane of the cochlea.