鼠脑听觉中枢在听毛细胞受损后的神经可塑性改变及毛细胞的损伤修复研究

Hair cells can repair after hearing impairment in non-mammals such as birds. However, such renovation is subatantially lacking in mammals. The present study is designed to explore the mechanism of the above difference,and to find out the genes which are involved in the repair after hearing impairment.Our study is valuable to find clues to cure the related diseases of human hearing. It includes the following objectives: 1）To find out the changes of the neural plasticity of auditory centers in the rat brain after hearing impairment, including the changes of the cell morphology and cell number due to apoptosis, the neurotransmitters and their receptors, axon reorganization, and ultrastructure. Meanwhile, we also planned to examin the neural plasiticity of auditory nuclei in the quial brain, including how newborn cells are produced,differentiated,and integrated into the existed neural pathways. 2)To determine which genes are involved in hair cells repair and what roles they play after hearing impairment. The genes which are related to the differences of hair cells repair after hearing impairment between mammals and non-mammals could be determined through the differences of their expression during hair cells repair between mammals and non-mammals.These genes will be further taken into the inner ear by Lentiviral vector ( LVs) to know whether they could improve hair cells repair. To evaluate experimental animal hair cell function，the click-evoked potentials on the brain stem (auditory brainstem response, ABR) will be recorded and neural plasticity will be examined as described above.Our study will provide useful guides to the treatment of deafened people by gene therapy.

哺乳动物听毛细胞与神经细胞一样，一旦受损则不能被替换或修复，从而可导致人永久性耳聋。新的统计表明我国听力障碍患者位于各类残疾之首。听感觉毛细胞死亡后，听力丧失患者脑内听觉中枢发生了哪些神经可塑性变化？目前依然不清楚。而许多报道表明：非哺乳动物如鸟类内耳听毛细胞损伤后可以完全修复。为此，本项目拟先对鼠内耳毛细胞受损后，研究其听觉脑中枢（脑桥蜗神经核、中脑下丘、间脑内侧膝状体）发生了哪些神经可塑性变化（细胞凋亡、神经递质及其受体、轴突生长发生、超微结构等），并同时弄清鸟毛细胞损伤修复中，出现了哪些可塑性变化，尤其关注新生细胞如何增殖、分化以及整合到已有神经通路中等。之后，我们希望通过哺乳动物（鼠）和非哺乳动物（如鸟）听感觉毛细胞损伤后修复过程中相关基因的表达差异，利用病毒载体基因导入技术，找出影响哺乳动物听感觉毛细胞损伤修复能力最大的基因或基因组合。为人类听力疾患的治疗基因提供有用数据。

听感觉毛细胞（Hair cell，HC）是接收声音振动并转化为电信号的基本元件。哺乳动物中的HC由于药物、噪音等原因损伤后不能自我修复。然而，在鸟类等非哺乳动物中，HC在损伤后可以再生并恢复功能，这便为哺乳动物HC再生带来了希望。本实验以鸡和小鼠为研究对象，构建损伤模型。结果发现，庆大霉素可以进入E12鸡体内及HC，高浓度（75、150及250mg/kg）庆大霉素能使鸡胚孵化率显著降低，但HC并不受损伤，其中可能存在某种保护机制使HC不受伤害；新生雏鸡庆大霉素处理后，HC从近端向中端损伤，损伤部位的支持细胞（Supporting cell，SC）出现增殖，并通过有丝分裂和转分化方式再生HC；小鼠耳蜗体外培养体系中，正常培养条件下HC未出现损伤，庆大霉素处理后HC从近端至远端逐渐损伤，而且随着培养时间的延长损伤程度不断扩大。对PG2d（增殖起始）和PG3d（增殖高峰）小鸡耳蜗上皮及PG3d小鼠耳蜗上皮分别进行转录组分析。雏鸡耳蜗上皮中检测到16587个基因，PG2d起始增殖时649个基因和104个信号通路发生变化，PG3d增殖高峰期时805个基因和99个信号通路发生变化；变化的通路中都包含了与HC发育或增殖相关的通路Notch、Wnt、Hh、MAPK、TGF-β信号通路及cell cycle，除了Hh通路，这些信号通路都在前50通路中。小鼠耳蜗上皮中检测到22086个基因，2243个基因的表达及265个信号通路发生变化，包括HC发育或增殖相关的通路Notch、Wnt、Hh、MAPK、TGF-β信号通路及cell cycle，但除了MAPK通路，其他通路都不在前50中，并不是小鼠HC损伤后的主要细胞活动。抑制Notch信号通路能诱导小鼠SC的增殖，并且能通过转分化和有丝分裂两种方式产生新HC，以转分化为主；Licl增强Wnt信号对HC有保护作用，但没有促进细胞再生；BIO增强Wnt信号通路及SAG增强Hh信号对HC没有影响；DAPT/Licl同时抑制Notch信号和增强Wnt信号可通过转分化促进HC再生；DAPT/BIO抑制Notch信号和增强Wnt信号对HC没有影响；DAPT/SAG抑制Notch信号和增强Hh信号对HC则加大了HC的损伤程度；同时调控两种通路消减了单独调控Notch通路对HC的影响。综上，本实验为从转录组调控角度为小鼠耳蜗HC不再生原因提供了依据.