声刺激调控Clusterin表达在大鼠脑干耳蜗核神经干细胞增殖分化中的作用及机制研究

Activating and keeping the proliferation and differentiation of neural stem cells in hearing system of sensorineural deafness treatment is of great significance. The previous studies found that the expression of Clusterin affect the various factors inducing cell apoptosis and differentiation. Also, Clusterin regulated mitochondrial function. Our previous studies show that (1) there are neural stem cells (NSCs) in the cochlear nuclei of the rat brainstem; (2) there is a specific relationship between the changes of Clusterin expression and acoustic stimulation in neural stem cells of rat brainstem cochlear nuclei; (3) the expression of Clusterin affect cell apoptosis and differentiation; (4) Acoustic stimulation influences the mitochondria. The previous results strongly suggested that Acoustic stimulation regulation the expression of Clusterin to affect proliferation and differentiation of neural stem cells (NSCs) in the cochlear nuclei of the rat brainstem may be associated with mitochondrial function change. This project is aimed to investigate the expression of different Clusterin subtypes in newborn rat's cochlear nucleus under acoustic stimulation, and the effect of knocking down of Clusterin by using RNAi and TALENs on the proliferation, differentiation and mitochondria function of neural stem cells in newborn rat's cochlear nuclear. This project provides new experimental basis in the research on auditory development, and the remodeling of auditory nerve system structural and functional after injury.

激活并保持听觉系统自身神经干细胞的增殖分化能力对感音神经性耳聋的治疗具有十分重要的作用。新近研究发现，Clusterin的表达变化能够影响各种因素诱导的细胞凋亡和分化状态。研究提示，Clusterin可调控线粒体功能。前期工作显示：①新生大鼠脑干耳蜗核中存在神经干细胞；②脑干耳蜗核神经干细胞Clusterin的表达与声刺激存在特异性联系；③Clusterin表达影响细胞凋亡与分化；④声刺激能够影响线粒体的功能。因此推测：声刺激调控Clusterin的表达对大鼠脑干耳蜗核神经干细胞增殖分化产生影响可能与线粒体功能改变相关。本项目拟在声刺激条件下检测Clusterin不同亚型在新生大鼠脑干耳蜗核表达的变化，并且采用RNAi、TALENs等技术阻断Clusterin亚型的表达研究神经干细胞增殖分化和线粒体功能的变化。以期为听觉发育研究及听觉神经系统损伤后的结构和功能重塑提供理论和实验依据。

感音神经性耳聋已成为常见的引起听力功能障碍以至致残的重要原因。哺乳动物听觉中枢神经核团中存在神经干细胞（NSCs），该细胞增殖、分化、迁移与外界声刺激密切相关。如何保持听觉系统内源性NSCs的“干”性，是重建神经网络、修复听觉功能的关键。线粒体对于维持干细胞的增殖、分化和更新有关键作用。神经调节因子Neuregulin-1（Nrg1）能与受体酪氨酸蛋白激酶家族的成员ErbB结合，诱导广泛的生物学功能。但Nrg1/ErbB2在耳蜗神经核NSCs发育和分化中的功能及其在调控线粒体动力学和生物产能体系中的作用尚未明确。本项目研究了Nrg1/ErbB2对新生大鼠脑干耳蜗核NSCs分化和凋亡、线粒体动力学和产能学的影响以及对下游PI3K/Akt信号通路的影响，发现大鼠耳蜗核NSCs中NGR1和ErbB2的表达随着年龄的增加而增加。NRG1处理NSCs能减少nestin阳性细胞，增加MAP2阳性和GFAP阳性细胞，降低细胞凋亡，增加PI3K/AKT活化。PI3K/AKT抑制剂LY294002处理细胞可增强ErbB2沉默对nestin、MAP2、GFAP和细胞凋亡的影响。NRG1处理NSCs能促进线粒体分裂蛋白(Mfn) 1和Mfn2的表达，降低线粒体裂变蛋白1 (Fis1)和动力相关蛋白1 (Drp1)的表达。NRG1处理细胞后，细胞氧化氢酶、SOD、GSH-Px活性升高，过氧化氢酶、MnSOD、PGC-1α、NRF1和TFAM的蛋白表达水平升高。敲除ErbB2后，能部分逆转NRG1诱导的mtDNA拷贝数和细胞内ATP水平升高、ATP酶活性升高以及乳酸生成降低。本课题的完成阐明了激活“唤醒”听觉系统内源性干细胞增殖分化的机理，为开展听觉系统神经干细胞移植、听觉发育研究及听觉神经系统损伤后的结构和功能重塑等提供了实验依据。