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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 study the feasibility of adenovirus carrying gene transduction through cochlear via micro-injection of round window membrane in mouse ear and to provide experimental basis and anatomic evidence for gene therapy of inner ear in mice. Methods Twelve C57BL / 6J mice were divided into two groups. The experimental group (n = 8) received recombinant adenovirus carrying enhanced green fluorescent protein (EGFP) and the control group (n = 4) Liquid through the ear into the round window membrane microinjection of cochlear injection. Bilateral cochlea specimens were taken as basement membrane on day 5 and day 14, respectively. GFP expression was observed under confocal laser scanning microscope. Results Postoperative animals survived 10 (1 death per group). After transfection in the experimental group, the gene expression on the basement membrane of the basal cochlea and the target gene on the spiral ganglion was significant, which was stronger in the 14-day group than in the 5-day group. The control group showed no fluorescence expression in the cochlea. Conclusion The posterior approach is simple, less invasive and easy to expose the round window niche. The method of transducing the target gene by microscopic injection of the round window membrane into the posterior ear pathways can successfully transduce the gene of interest into the cochlear tissue and express it.