调控组蛋白H3K9甲基化状态促进哺乳动物耳蜗毛细胞再生

Histone H3 Lysine9 methylation plays a critical role in embryonic development and stem cell differentiation. Our previous study showed that dimethylation of lysine 9 on histone H3 (H3K9me2) involved in development of zebrafish neuromast and hair cell proliferation, and BIX01294,a small-molecule inhibitor for the G9a Histone methyltransferase promote progenitor cells proliferation and hair cell regeneration in the neuromast of zebrafish. The role of H3K9 methylation in the developing mammalian cochlea is still unclear. In this project, we will investigate the spatial and temporal pattern of H3K9 methylation in the developing mouse cochlea and its role of hair cell regeneration. Immunofluorescence and LCM-WB analysis will be used to detect the expression of H3K9 methylation in the developing mouse cochlea; To initiate progenitor cells proliferation and promote hair cell differentiation and regeneration by regulation of Histone H3 Lysine9 methylation, auditory epithelium dissected from new born mouse cochlear will be treated with neomycin first during tissue culture in vitro , and after hair cell damaged, treated with BIX01294 secondly. That will be a novel strategy of sensorineural deafness treatment by a small molecule compound.

H3K9(组蛋白H3第9位赖氨酸)甲基化状态在胚胎发育和干细胞分化过程中发挥重要作用。我们已有的研究显示，H3K9甲基化参与斑马鱼神经丘发育过程中毛细胞的增殖分化。应用BIX 01294（组蛋白甲基转移酶G9a拮抗剂）能够促进斑马鱼神经丘前体细胞增殖和毛细胞再生。组蛋白H3K9甲基化在哺乳动物耳蜗发育中的作用尚无报导。本项目拟研究H3K9甲基化水平在小鼠内耳不同发育阶段的时空表达模式及其在哺乳动物耳蜗毛细胞再生中的作用。拟采用免疫荧光和LCM-WB方法观察H3K9甲基化在小鼠耳蜗发育过程中的时空表达模式；采用新生小鼠耳蜗新霉素毛细胞损伤模型，在听感觉上皮体外培养组织中应用BIX 01294调控H3K9甲基化状态，试图激活耳蜗前体细胞增殖，促进毛细胞分化和毛细胞再生，以期修复损伤的听觉功能，为临床治疗感音神经性聋提供新的策略。

斑马鱼侧线器神经丘支持细胞的活化再生致毛细胞再生。斑马鱼侧线器毛细胞再生的调节机制中表观遗传学因素已见报道，但H3K9me2在毛细胞损伤后再生的调节机制尚不明了。本研究中，我们旨在阐明新霉素损伤介导的毛细胞损伤后再生过程中H3K9me2的作用。BIX-01294，G9a/GLP特异性抑制剂，能显著抑制H3K9的二甲基化。我们发现BIX-01294显著减少新霉素介导的毛细胞损伤后的再生，BIX同时减少了神经丘细胞的增殖致支持细胞数减少。原位杂交试验表明BIX01294显著下调Wnt和Fgf信号通路，从而导致支持细胞增殖及毛细胞再生的减少。总之，我们的结果表明下调H3K9me2使Wnt/β-catenin和Fgf信号通路失活，从而抑制了新霉素介导的毛细胞损伤后的再生。H3K9me2在毛细胞再生过程中起到了重要作用，作为一个新型的生物标志物，亦为治疗听力损伤的潜在治疗作用靶标。.在本研究的哺乳动物实验部分，我们已经获得了小鼠耳蜗基底膜发育过程中H3K9me2的表达模式数据，以及调控组蛋白甲基化的初步数据。