Math1基因内耳导入径路的探索研究

目的研究腺病毒携带Math1-EGFP基因经完整圆窗膜途径及鼓阶打孔途径导入耳蜗后对听功能和转导效率的影响，为内耳基因治疗提供实验基础和理论依据。方法健康成年白色红目豚鼠40只，雌雄不限，体重250—300g。随机分成四组，完整圆窗膜组12只，鼓阶打孔组12只,各组分别设对照8只。实验组（24只）导入重组腺病毒携带的Math1基因及增强型绿色荧光蛋白基因（enhanced green fluorescent protein，EGFP），对照组（16只）导入人工外淋巴液，所有动物均以左耳作为导入耳。术前及术后分别行听性脑干反应（ABR）检查。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片观察基因表达情况。结果完整圆窗膜组导入耳ABR阈值，术后5天各频率与术前比较无显著性差异（P〉0．05）；鼓阶打孔组导入耳ABR阈值，术后5天在2kHz、4kHz与术前比较无差异（P〉0．05），8kHz较术前增高（P〈0．05），16kHz、20kHz较术前明显增高（P〈0．01），术后14天在16kHz、20kHz较术后5天时明显好转（P〈0．01），但较术前仍有增高（P〈0．05）。转导成功率鼓阶打孔组为91．6％,优于完整圆窗膜组的50％。两种转导途径对目的基因在耳蜗内的表达部位和表达时间没有显著影响。结论完整圆窗膜途径及鼓阶打孔途径在转导成功率及听功能保护方面各有优劣。完整圆窗膜途径因其对耳蜗的损伤极小，在临床应用方面具有更好的发展前景。

The surgical approach of Math1 gene transfer into cochlea in the guinea pig

Objective To study effects of the transduction efficiency of adenoviral-mediated Math1-EGFP gene in the guinea pig cochlea after transfer through intact round window membrane (RWM) or via the scala tympani (ST),and to assess their influence on hearing and to provid experiment foundation for inner ear gene therapy.Methods Forty adult guinea pigs were used,with no sex limited and weight between 250 and 300g.The animals were divided into four groups in random,12 were surgically inoculated with Ad-Math1-EGFP in the bony groove of round window niche,12 for inoculated with Ad-Math1-EGFP via the scala tympani,and 8 with artificial perilymph as control group for each of the experimental group.All the animals were treated on left ear.Auditory brainstem response (ABR) thresholds were determined in all animals before and after surgery.On post-surgery day 5 and day 14,animals were sacrificed and whole mounts of cochlea and frozen sections were examined.Results ABR threshold showed no significant change at 5 days after the surgery in group RWM (p>0.05),as well as the group ST at 2 kHz and 4 kHz,while ABR threshold showed significant change at 8 kHz (P<0.05) and at 16kHz & 20 kHz(P<0.01) in the group ST.In 14 days after the surgery,the group ST became better at 16kHz & 20kHz (P<0.01),but showed no significant change of heating compare with the surgery before (p>0.05).Transduction efficiency of the group ST is 91.6%,better than that of the group with RWM (50%).There was no significant difference in the location and time of the expression of Mathl-EGFP gene in the guinea pig cochlea between two groups.The contralateral cochlea and those in the control groups were free of fluorescence staining.Conclusion Transfering through intact round window membrane or via the scala tympani has different effect on innerear cells.Transfer through intact round window membrane has less damage to cochlea,with maybe has a better future in clinical application.