鼓室内显微注射慢病毒转染小鼠耳蜗的技术改良

Technical Improvement of Lentivirus Transfection into Mouse Cochlea by Intratympanic Microinjection

目的改良经鼓室内显微注射慢病毒转染小鼠耳蜗的实验技术。方法将30只BALB/c小鼠随机分为3组:1μl/min组(12只)和20μl/min组(12只)小鼠经腹侧入路鼓室内分别以1μl/min和20μl/min的速度显微注射5μl的携带绿色荧光蛋白基因的慢病毒(LV3-GFP),人工外淋巴液组(6只)以1μl/min速度注入等量外淋巴液。于手术前、术后1、3、7天各组动物行听性脑干反应(ABR)测试后,进行耳蜗基底膜铺片荧光染色和耳蜗切片免疫荧光染色,并在激光共聚焦显微镜下观察慢病毒转染情况。结果各组小鼠术后1天ABR阈值均较术前明显升高(P<0.01),1μl/min组和人工外淋巴液组术后3天ABR阈值恢复正常,20μl/min组术后7天恢复正常。慢病毒转染后,1μl/min组术后1天在耳蜗毛细胞观察到慢病毒阳性表达,术后3天在耳蜗Corti器、螺旋神经节及血管纹处均可见阳性表达;术后1、3、7天耳蜗中回外毛细胞转染率平均为98.3%、98.1%和98.7%,而20μl/min组术后1天未见明显阳性表达,术后1、3、7天耳蜗中回外毛细胞的转染率分别为4.2%、20.2%和47.3%。结论鼓室内显微慢速注射法可将携带目的基因的慢病毒高效转染至耳蜗组织并表达。

Objective To improve the experimental technique of transferring the lentivirus carrying green fluorescent protein(LV3-GFP) into mouse cochlea by intratympanic microinjection. Methods Thirty BALB/c mice were randomly divided into three groups: 1 μl/min experimental group(12 mice) and 20 μl/min experimental group(12 mice) were received 5 μl intratympanic microinjection to carry LV3-GFP at different speeds(1 μl/min or 20 μl/min)through the anterior ear path, respectively, and the artificial perilymph group(6 mice) was injected with the same amount of perilymph by 1 μl/min speed. Auditory brainstem response(ABR) test were used to observe at 1 day before operation, postoperative day 1, 3 and 7, respectively. Then the organ of Corti was dissected from the cochlea, stained with TRITC phalloidin to count the average number of normal hair cells in each turn of the cochlea. And immunofluorescence staining was employed to localize LV3-GFP in cochlear slices. Laser confocal microscopy was used to observe the location and efficiency of transfection. Results In all groups, ABR threshold of mice was higher on the first day after operation than that of before operation(P<0.01). In 1 μl/min experimental and artificial perilymph group, ABR threshold returned to normal level on the third day after operation(P>0.05), while in the 20 μl/min experimental group, it was not restored(P<0.01), suggesting that the modified injection method was less harmful to the auditory function of mice. In 1 μl/min exprimental group, after lentivirus transfection, positive expression was found in the cochlear hair cells on the first day after the operation, and it was also found in the Cort organ, spiral ganglion and stria vascularis of the inner ear from a mouse on the third day after the operation. In 1 μl/min experimental group, on the frist day after operation, the average transfection efficiency was 98.3%, 98.1% and 98.7% in the middle turns of basement membrane, respectively. There was no significant positive expression at 1 day after operation, and the transfection efficiency at 1, 3 and 7 days after operation was 4.2 %, 20.2% and 47.3%, respectively. Conclusion The result show intratympanic microinjection method can efficiently transfer the lentivirus carrying the target gene into the cochlea and express it.