Notch信号通路在树鼩耳蜗感觉前体细胞增殖和分化中的调控作用

Damage to hair cells in adult mammals can causes permanent hearing impairment because these cells can’t regenerate. In recent years, the sensory progenitor cells can re-enter the cell cycle and regenerate into hair cells in the neonatal cochea by activing Wnt and Noctch related signaling pathways. the study of hearing recovery have become a hot spot study. However, little research in primates. in these years, our team has been engaging in the studies of the auditory system in tree shrew of primate. we found that the cochlear support cells have proliferation ability by activating the Wnt signaling pathway. On the basis of preliminary studies,we will study the regulation of Notch signaling pathway in the proliferation and differentiation of the cochlear precursor cells in the tree shrew, including the expression of Notch signaling pathway in different age groups of cochlea in tree shrews; the expression of Notch signaling pathway in the cochlea after using Neomycin; culture tree shrew basilar membrane in vitro, observe the regulation of Notch signaling pathway in the proliferation and differentiation of the cochlear precursor cells in the tree shrew before and after using Neomycin. The purpose of this study is to elucidate the role of Notch pathway in the regeneration of cochlear hair cells. It provides an experimental basis for the in-depth study of the complex network relationship between the signal pathways of the cochlear hair cell regeneration and the precise regulation of the cell regulatory factors.

成年哺乳动物听觉毛细胞损伤后导致不可逆性耳聋。研究发现：调控Wnt、Notch等信号通路、诱导耳蜗前体细胞重新进入细胞周期，增殖分化为毛细胞使听力功能恢复的研究成为热点。然而目前该研究在灵长类动物中无针对性报道。本课题组前期致力于树鼩外周听器的研究，发现激活Wnt信号通路后耳蜗支持细胞具有增殖能力。本项目拟在此基础上开展研究Notch信号通路在树鼩耳蜗中前体细胞中的增殖和分化中的调控作用。具体包括：Notch信号通路在不同年龄组树鼩耳蜗中的表达；新霉素破坏成年树鼩耳蜗毛细胞后，Notch信号通路相关指标在耳蜗中的表达；体外培养新生树鼩耳蜗基底膜，通过激活或抑制Notch通路，观察Notch信号通路在新霉素损伤毛细胞前后的耳蜗前体细胞増殖、分化中的调控作用。通过上述研究力图阐明Notch通路在耳蜗毛细胞再生中的作用，为耳蜗毛细胞再生的各信号通路之间精确调控的复杂网络关系的深入研究提供基础。

在前期研究中我们已对灵长类动物树鼩的外周听觉器官的解剖结构、Micro-CT等进行了研究，并通过树鼩的耳蜗基底膜进行体外培养，发现激活Wnt信号通路后耳蜗支持细胞具有增殖能力。本次课题在前期研究的基础上，进一步研究了树鼩内耳的超微结构，以及成功建立了庆大霉素损伤树鼩内耳的模型，通过转录组学比较分析，筛选出树鼩正常耳蜗组织与庆大霉素耳毒性耳蜗组织的差异表达基因，结果发现：多条信号途径介导庆大霉素对树鼩耳蜗的损伤。其中 MAPK 信号通路、JAK-STAT 信号通路等均出现明显的差异性表现。在体外研究中，已完成对小鼠Notch信号通路γ-分泌酶抑制剂LY3039478调控小鼠耳蜗前体细胞增殖分化和毛细胞再生的作用分析。结果发现：Notch信号通路γ-分泌酶抑制剂LY3039478协同WNT信号通路激动剂CHIR-99021对小鼠耳蜗前体细胞增殖分化和毛细胞再生起促进作用。通过转录组学比较分析，发现DMSO对照组及CHIR+LY联合组信号通路呈现差异性基因变化。DMSO对照组及CHIR+LY联合组差异表达基因共有981个，其中上调200个，下调781个；我们关注的wnt通路有17个差异基因Lgr5/Frat1/Znrf3/Serpinf1/Fzd9/Daam2/Frat2/Wnt6/Rspo1/Rac2/Dkk2/Wnt4/Ccnd1/Wnt10a/Wnt10b/Sfrp1/Mapk10发生变化和Notch通路4个差异基因Lfng/Jag1/Dll3/Dtx3l表达发生变化，经过qRT-PCR 检测Wnt6，Atoh1等基因的表达趋势与转录组测序结果基本一致。在庆大霉素损伤毛细胞的体外研究中，LY3039478具有保护毛细胞的作用。通过本课题的研究，为耳蜗毛细胞再生的各信号通路之间精确调控的复杂网络关系的深入研究提供基础。