经圆窗膜鼓阶显微注射腺相关病毒转染小鼠耳蜗的研究

The Study of Adeno-associated Virus Transfection into Mouse Cochlea by Scala Tympani Microinjection via the Round Window Membrane

目的探讨经圆窗膜鼓阶显微注射携带绿色荧光蛋白基因的重组腺相关病毒AAV2/9-GFP转染小鼠耳蜗的可行性,为应用腺相关病毒治疗遗传性聋提供实验基础。方法将24只野生型C57BL/6J小鼠随机分为三组:实验组(12只)和人工外淋巴液组(6只)分别经耳后径路通过圆窗膜向鼓阶内注射2μl AAV2/9-GFP或人工外淋巴液;空白对照组(6只)正常饲养,不做任何处理。于术后21 d进行ABR测试,之后取耳蜗基底膜行免疫荧光染色,激光共聚焦显微镜观察。结果术后实验组和人工外淋巴液组术侧耳ABR各频率反应阈均不同程度升高,与空白对照组比较差异均有统计学意义(P<0.05),实验组与人工外淋巴液组比较差异无统计学意义(P>0.05)。实验组转染AAV2/9-GFP后可见基底膜底回至顶回均有GFP表达,转染阳性细胞呈逐渐减少的趋势,底回、中回及顶回的内毛细胞平均转染效率分别为82.7%、75.0%、44.7%,底回与顶回、中回与顶回转染效率比较差异均有统计学意义(P<0.05),底回与中回比较差异无统计学意义(P>0.05)。AAV2/9-GFP主要转染内毛细胞,外毛细胞未见GFP表达;实验组对侧耳、人工外淋巴液组及空白对照组均未见GFP表达。结论携带目的基因的腺相关病毒载体可通过经圆窗膜鼓阶显微注射的方法转染至小鼠耳蜗内毛细胞并表达。

Objective To investigate the feasibility of adeno-associated virus carrying green fluorescence protein gene (AAV2/9-GFP)transfection into mouse cochlea by scala tympani microinjection via round window membrane. Methods A total of 24 wild type C57BL/6J mice were divided into three groups. Animals in the experimental group (12 animals) and the artificial perilymphatic fluid (APF) group (6 animals) were microinjected with 2 μl AAV2/9-GFP and APF respectively into scala tympani via round window membrane by postauricular approach while the animals in the control group (6 animals) were kept untreated. ABR test was carried out in all groups 21d after the operation. Then all the animals were sacrificed and cochleas were harvested for immunofluorescence. Results The ABR thresholds of the operative ears of the animals in the experimental group and the APF group were significantly elevated compared with the control group after the operation ( P < 0.05 ). There were no significant differences between the experimental group and the APF group ( P > 0.05 ). GFP expression was observed in the experimental group. The green fluorescence positive cells gradually decreased from the basal turn to the apical turn of the cochlear basal membrane. The transfection efficiency of the inner hair cells of the basal, middle and apical turn was 82.7%, 75.0% and 44.7%, respectively. The difference of transfection efficiency was significant between the basal and apical turn, middle and apical turn ( P < 0.05 ), but there were no significant differences between the basal and middle turn ( P > 0.05 ). AAV2/9-GFP mainly transfected inner hair cells. No GFP expression was observed in outer hair cells. There was no GFP expression in the contralateral ears of the experimental group, the ears of the APF group nor in the control group. Conclusion Adeno-associated virus carrying the target genes can successfully transfect mouse cochlear inner hair cells by scala tympani microinjection via round window membrane.