功能性耳蜗螺旋神经元细胞再生研究

哺乳动物耳蜗神经元细胞自然状态下一旦损伤，无法自我更新修复。目前听力治疗主要借助人工耳蜗和助听器的使用，而治疗效果往往取决于螺旋神经元细胞是否具有功能，如何修复受损伤的神经元是听觉领域的重要课题。申请人之前的研究表明耳蜗胶质细胞过表达Neurog1和Neurod1，能转分化为新生的神经元细胞，但新生神经元细胞未分化成熟。本项目将进一步探索影响新生神经元分化成熟的因素。研究策略是：1）构建耳蜗神经元细胞损伤小鼠模型，研究损伤对胶质细胞转分化神经元细胞产生的影响；2）分析比对实验室已建立的耳蜗螺旋神经元细胞RNA-Seq数据库，利用小鼠基因体内快速敲除方法筛选出调控神经元细胞分化成熟的关键基因；3）建立关键基因的条件性过表达小鼠模型，与Neurog1﹑Neurod1同时在胶质细胞中过表达，检测能否再生具有功能的新生神经元细胞。本项研究能够为听觉损伤的修复提供重要的理论依据，具有现实的临床意义。

Impairments of spiral ganglion neurons (SGNs) in mammal cochleae will never self-regenerate and restore under the natural conditions, thereby resulting in the permanent deafness. Fortunately, it can be rescued via medical devices, such as hearing aids and cochlear implants. But their success critically depends on having functional SGNs cells to relay auditory information to the brain. It is of clinical importance to investigate how to regenerate spiral ganglion neurons. The previous studies of applicant support that ectopic expression of SGNs specific gene Neurog1 and Neurod1 convert glia cells into SGNs. Unfortunately, the newly generated SGNs are not fully differentiated. Our project will focus on identifying the key gene families that control the entire differentiation program of SGNs. Our strategies are divided into three steps: 1) we will generate Lypd1-DTR/+ mice and test whether SGNs will be killed after injected Diphtheria toxin (DT). Furthermore, we will detect whether the SGN death affect Neurog1 and Neurod1 mediated glia to SGN fate conversion；2）we will choose the most dynamic genes as candidates from our RNA-Seq data to perform further functional tests via CRISPR/Cas9 knock out approach; 3）we will kill the original SGNs by injecting DT, and then conditionally overexpress the key gene、Neurog1 and Neurod1 to furtherly check whether newborn functional SGNs will be generated via immunohistochemical staining and ABR test. Taken together, our proposed study will provide foundations for how to use glia cells to regenerate new functional SGNs in clinic.