耳后入路圆窗膜显微注射小鼠耳蜗基因转染新途径的研究

The Study of a New Approach to Postauricular Microinjection via the Round Window Membrane for Cochlear Gene Transfection in Mouse

目的研究腺病毒携带目的基因经小鼠耳后入路圆窗膜显微注射途径耳蜗转导的可行性,为以小鼠作为动物模型的内耳基因治疗提供实验基础和解剖学依据。方法12只C57BL/6J小鼠分为2组,实验组(8只)以重组腺病毒携带的增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)、对照组(4只)以人工外淋巴液经耳后入路圆窗膜显微注射注入耳蜗内。分别于术后5、14天取双侧耳蜗标本做基底膜铺片,在激光共聚焦显微镜下观察GFP表达。结果术后动物存活10只(每组死亡1只)。实验组转染后耳蜗底回基底膜及螺旋神经节上目的基因有表达,14天组强于5天组。对照组耳蜗未见荧光表达。结论耳后入路操作简单、损伤小、易于暴露圆窗龛。耳后入路圆窗膜显微注射腺病毒携带目的基因转导的方法能够将目的基因成功转导至耳蜗组织并表达。

Objective To assess the feasibility of adenoviral vectors mediate cochlear gene transfer by postau- ricular microinjection through the round window membrane in mouse. Methods Twelve 5--week old C57BL/6J mice were selected for the study： 8 were implanted with Ad--EGFP by postauricular microinjection through the round window membrane, and 4 with artificial perilymphatic fluid. On postoperative days 5 and 14, the animals were sac- rificed and the surface preparation of cochleae was observed. Results Two animals died after operation. Bright green fluorescence in the cochleae was observed in Ad--EGFP groups. Gene expression on day 14 after operation was higher than that on day 5. However, the control group was free of fluorescence. Oonclusion The postauricular route of the cochlear gene transfer in mice is simple to operate with little side-- effect. The technique of transgenic delivery into the inner ear through RWM bv microiniection is feasible and effective.