光遗传学通道（ChR2）对螺旋神经节细胞兴奋性调控机制的研究

Hearing loss is a serious disease that affects people's life. At present, Cochlear implant is an efficient therapeutic way for the heavily or profound hearing loss which would promote the formation of human auditory via stimulating the auditory nerve directly. Due to the high cost of the of cochlear implant which will aggravate the economic burden to patients and society , low cost and long efficiency alternative method need to be found. Optogenetics channel (ChR2) is widely used in the activity of neural cells in the brain. There is still not enough evidence which is proved that ChR2 is expressed and exhibits functions in spiral ganglion cells (SGNs) in the cochlea. Here, we present a novel strategy to transfect the remanent SGNs by ChR2 which is carried by various vectors. Optogenetics channel (ChR2) posses the characteristics of optical-eletrical switch that will promote acoustic signals into the nerve impulses. Finally, the auditory system would transduce the signals to the auditory cortex which account for the formation of mammalian hearing. The exacting regulation mechanism of Optogenetics (ChR2) on SGNs not only paved the way for the underlying mechanism of hearing loss and clinical therapy but also improved the health level and quality of life for people suffering hearing loss.

耳聋严重影响人们的生活。目前重度、极重度耳聋有效治疗手段是植入人工耳蜗，直接刺激听神经形成人工听觉。但人工耳蜗价格高昂，给患者及社会带来巨大经济负担，亟需寻找成本低廉、长效的替代方法。光遗传学通道（ChR2）被广泛应用于脑细胞的神经活动研究。目前ChR2通道在耳蜗螺旋神经节细胞中的表达及功能调控等方面仍缺乏直接实验依据。本研究拟将光敏感通道蛋白（ChR2）基因通过多种载体导入内耳残存螺旋神经节细胞，利用光敏感蛋白具有的光电换能特性，实现音频能量向神经电信号的转换，并通过听觉传导通路将冲动传至听觉皮层从而产生听觉。以光遗传学通道（ChR2）对螺旋神经节细胞兴奋性调控机制的研究入手，可以为耳聋的发生机制及临床治疗提供新理论依据和思路，对提高国民健康水平、改善耳聋人群的生活质量具有重要意义。

本项目圆满完成了课题计划书列出的研究目标，分别从听觉功能、细胞生理与蛋白分子等多个层面上明确了光敏感通道蛋白（ChR2）在耳蜗螺旋神经节细胞上表达及功能调控作用。研究内容主要是针对缺乏对成年的动物模型的现况，通过转基因技术构建光遗传学的动物模型，寻找到了安全有效的转染途径构建内耳转染ChR2基因的动物模型，多种方法验证了病毒载体plenti-hSyn-hChR2(H134R)-EYFP与AAV-hSyn-hChR2(H134R)-EYFP构建成功；实验证明慢病毒转染基底膜的最佳滴度为6.25x105 PV；病毒转染后离体培养的基底膜可通过膜片钳记录到经470nm蓝光诱发的动作电位。利用自制导管圆窗导入病毒的方式听力损失小，大大提高了活体转染成功率。通过ChR2基因内耳转染的在体实验，成功建立了稳定可靠的成年动物模型，在国内首次成功记录到470nm蓝色激光诱发的耳蜗电位，实验发现470nm蓝色激光诱发的动物活体耳蜗电位与激光强度具有非线性相关的特性，同时进一步分析了激光照射的最佳理论照射强度为3.70 mW，可达到有效刺激的同时，减少对组织的损伤。本研究为应用光遗传技术治疗重度耳聋和极重度耳聋提供了新的实验依据。基于数据获得国家发明专利2项，公示1项，发表科研论文21篇，杂志接受3篇，参会2篇，所有文章均注明受自然基金资助。通过开展本项目培养博士研究生1名，硕士研究生6名。获得国家科技进步奖二等奖一项。