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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。
Objective To study the effect of adenovirus carrying Math1-EGFP gene on hearing function and transduction efficiency after intact round window membrane and cochlear implants, so as to provide experimental basis and theoretical basis for gene therapy of inner ear. Methods Healthy adult white red guinea pigs 40, male or female, weight 250 ~ 300g. Randomly divided into four groups, a complete round window membrane group 12, Drum perforated group 12, each group were set control 8. The experimental group (24 mice) was introduced with the recombinant adenovirus carrying Math1 gene and enhanced green fluorescent protein (EGFP), and the control group (16 mice) with artificial perilymph. All the animals were introduced with the left ear ear. Preoperative and postoperative auditory brainstem response (ABR) were examined. Bilateral cochlear specimens were taken at 5 days and 14 days postoperatively to observe the gene expression of the basement membrane. Results There was no significant difference in the frequency of ABR between the intact round window membrane and the ear on the 5th day after operation (P> 0.05) (P <0.05), but the difference between preoperative and postoperative was significant at 8 kHz (P <0.05). The preoperative values at 16 kHz and 20 kHz were significantly higher than those preoperatively (P <0.01) (P <0.01), but still higher than that before operation (P <0.05). The successful rate of transduction was 91.6% in the perforated group, which was better than 50% of the intact round window membrane group. Both transduction pathways had no significant effect on the expression site and expression time of the target gene in the cochlea. Conclusion The complete round window membrane approach and the multi-hole drilling approach have their own advantages and disadvantages in the success rate of transduction and the protection of hearing function. Because of its minimal damage to the cochlea, the complete round window membrane pathway has a better development prospect in clinical application.