Ad-GFP基因经半规管转导对豚鼠内耳形态和功能的影响

Influence and expression of GFP gene in the inner ears of guinea pigs by semicircular canal approach

目的:探讨经半规管途径转导豚鼠内耳基因的可行性,绿色荧光蛋白的表达及其对内耳形态和功能的影响。方法:采用白色红目健康纯种豚鼠24只,将缺陷型腺病毒携带的绿色荧光蛋白基因5μl经显微手术自前半规管灌入豚鼠内耳,同法注入人工外淋巴液5μl作对照,另一组经圆窗膜注入5μlAd-GFP基因,分别在手术后7、14、21、28d处死,0.5%硝酸银灌注耳蜗基底膜铺片显微镜观察内外毛细胞缺失,全耳蜗铺片荧光显微镜观察GFP表达。基因导入前后及处死前测定脑干诱发电位。结果:实验组耳蜗铺片内外毛细胞缺失计数和对照组无统计学意义;荧光显微镜下见术侧半规管、前庭、耳蜗均有荧光表达,术后7d表达最强,以后减弱,术后28d还有表达;经半规管组前庭区表达强于耳蜗区,主要分布在前庭及外淋巴间隙;经圆窗组耳蜗区表达强于前庭区。对侧耳蜗及对照组动物呈阴性表达。手术前后脑干诱发电位差异无显著性。结论:经半规管转导基因主要分布在前庭器官及外淋巴间隙,对耳蜗功能影响不大,是一种可行的途径;腺病毒载体携带的基因表达有一定的时间性。

Objective:To explore the feasibility and the expression of GFP gene in the inner ears of guinea pigs by semicircular canal approach,and to assess the disturbance to structure and function of the inner ear.Methods: Twenty-four white guinea pigs were used in this study.The auditory function was assessed by measuring the auditory brainstem respose(ABR).The experimental and control guinea pigs were inoculated with adenovirus carrying the reported gene GFP 5μl by the anterior semicircular canal approach or the round[HJ*2/5] window approach,and the control group was inoculated with artificial perilymphatic fluid 5μl.At 7 ,14 , 21 and 28 days, the animals were killed. The cochleas were stained with AgNO3 and the damaged hair cells of the coiti organ were counted. GFP′s expressions in the inner ears were observed with the fluorescent microscope. Results:The counts of the damaged hair cells had no statistical significance between the study group and the control group.The green fluorescence of the GFP gene expression was observed in the inner ears of the operative side,and the fluorescence reached a high level seven days after the adenovirus administration but reduced thereafter,while some was still observed at twenty-eight days. There were stronger expression near the inoculate position and the perilymphatic space in the transsemicircular canal group.While the stronger cochlears′ GFP expressions than the vestibulars′ were observed in the round window group.In the control animals and the inner ears,there was no expression. ABR thresholds had no significant difference before and after surgery.Conclusion: The GFP gene inoculated through the semicircular canal approach lies mostly in the vestibule and the perilymphatic space and causes little effect on the hearing threshold and the vestibular function.The gene carried adenovirus is limited expressed by time.