小鼠内耳圆窗基因导入的实验研究

Gene Delivery through Round Window Membrane in Mouse Inner Ear

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 expression of enhanced green fluorescence protein （EGFP） in inner ear of mice by microinjecting through the round window membrane with adenoviral vectors. Methods 19 healthy C57BL/6J mice were selected and divided into three groups： Ad--EGFP group, artificial perilymphatic fluid group and control group. 7 mice were microinjected Ad--EGFP through round window membrane, 6 mice were implanted with artificial perilymphatic fluid, and the left 6 mice were maintained as controls. Auditory brainstem response （ABR） thresholds were tested in all animals three days before surgery and seven days after surgery. All animals were sacrificed and the surface preparations of basilar membrane were carried out seven days after surgery. Results ABR results showed that this surgery had significant side--effect on the hearing of mice, but there was no signifi- cant difference between Ad--EGFP group and artificial perilymphatic fluid group after mircoinjection. Bright green fluorescence in cochleae was observed in Ad- EGFP group while artificial perilymphatic fluid group and control group were free of fluorescence. Conclusion The transgenic technique can successfully deliver EGFP into the inner ear by microinjection through round window membrane.