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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 investigate the feasibility of transduction of the inner ear gene of guinea pig by semi-regulatory route, the expression of green fluorescent protein and its effect on the morphology and function of the inner ear. Methods: Twenty-four white pure red healthy purebred guinea pigs were used. The defective adenovirus carrying 5μl of green fluorescent protein gene was injected into the inner ear of guinea pig from the first semicircular canal by microsurgery. In the same way, 5μl of Ad-GFP gene was injected through the round window membrane and sacrificed at 7, 14, 21 and 28 days after operation respectively. The defects of inner and outer hair cells were observed with 0.5% silver nitrate infusion cochlear basal membrane popcorn and the expression of GFP was observed with whole cochlear fluorescence microscope. Brainstem evoked potentials were measured before and after gene introduction and before sacrifice. Results: There was no significant difference between the experimental group and control group in the number of hair cells missing in the cochlear implants and in the control group. Fluorescence microscopy showed that the semicircular canal, vestibular and cochlear of the experimental group had the fluorescence expression, the expression was the strongest at 7d, and then decreased after 28d The expression in the vestibular area in the semicircular canal group was stronger than that in the cochlear area, mainly in the space between the vestibule and the perilymph. The expression in the cochlea was stronger in the cochlear area than in the vestibular area. Contralateral cochlear and control animals showed negative expression. There was no significant difference in brainstem evoked potentials before and after surgery. CONCLUSION: The trans-regulatory gene is mainly distributed in the space between vestibular organs and perilymph, which has little effect on the cochlear function. It is a feasible approach. The gene expression carried by adenoviral vector has a certain temporality.