协同调控Notch/Wnt信号通路对内耳干细胞增殖和分化潜能影响机制的研究

The irreversible damages on hair cells in the cochleae are the main reason for hearing loss. Since the inner ear stem cells could not automatically proliferate or transdifferentiate into other cell types for restoring the cochlear structure after the loss of hair cells, which significantly impedes the recovery of hearing. The investigation on the characteristics as well as the regulation on the biological behaviors of the inner ear stem cell will provide us the new strategies for activating inner ear stem cell for the regeneration of hair cell and following hearing recovery. We have demonstrated the interactions between Notch and Wnt signaling pathway during the development of the inner ear, the supporting cells could re-enter cell cycle and generate new hair cells after the co-regulation of Notch and Wnt signaling in the neonatal mouse cochlea. Since the supporting cells are composed of the several different subtypes, we will investigate the effects on the proliferation and trans-differentiation of the inner ear stem cell by co-regulating Notch and Wnt signaling specifically in the Lgr5+ supporting cells using transgenic mice. Afterwards, the regulated inner ear stem cells will be isolated for suspending proliferation and attached differentiation in culture medium, the RNA-Seq and ChIP-Seq with a small amount of stem cells at different stages will be applies for investigating the profiles of the genes expression as well as the epigenetic modification after the co-regulation of Notch and Wnt signaling. In order to determine the strategies for activating inner ear stem cell for the regeneration of hair cell, the important genes and epigenetic regulation sites will be selected for further verification through the 3D culture system of inner ear stem cells.

耳蜗毛细胞损伤将导致听觉障碍，哺乳动物耳蜗内干细胞长期处于静止状态，不能发挥干细胞特性修复受损的感觉上皮，为听觉功能的恢复带来了困难。研究耳蜗干细胞调控机制，将为激活干细胞促进毛细胞再生和听觉重建奠定基础。我们前期研究发现耳蜗发育中Notch和Wnt信号通路存在相互作用，协同调控Notch/Wnt信号通路能促进耳蜗支持细胞增殖和毛细胞再生。鉴于支持细胞的异质性，为特异性研究内耳干细胞调控机制，本研究利用转基因小鼠在Lgr5+内耳干细胞中协同调控Notch/Wnt信号，研究其对内耳干细胞增殖和分化的影响，同时利用纯化内耳干细胞体外扩增和贴壁分化模型，结合微量细胞RNA-Seq和ChIP-Seq技术研究协同调控Notch/Wnt影响内耳干细胞增殖和分化的机制，筛选调控干细胞生物学行为的关键基因和表观位点，结合内耳干细胞3D培养对调控的关键靶点进行验证，探讨激活内耳干细胞促进毛细胞再生的策略。

耳蜗毛细胞损伤将导致听觉障碍，哺乳动物耳蜗内干细胞长期处于静止状态，不能发挥干细胞特性修复受损的感觉上皮，为听觉功能的恢复带来了困难。研究耳蜗干细胞调控机制，将为激活干细胞促进毛细胞再生和听觉重建奠定基础。我们前期研究发现耳蜗发育中Notch和Wnt信号通路存在相互作用，协同调控Notch/Wnt信号通路能促进耳蜗支持细胞增殖和毛细胞再生，鉴于支持细胞的异质性，本研究进一步筛选了调控干细胞生物学行为的关键基因和表观位点，结合内耳干细胞3D培养对调控的关键靶点进行验证，探讨激活内耳干细胞促进毛细胞再生的策略。主要成果包括：1）利用小鼠内耳发育模型和微量细胞的RNA-Seq技术，发现Notch与Wnt信号通路在内耳感觉上皮命运决定以及毛细胞分化过程中的相互作用，进一步揭示内耳干细胞增殖和分化调控的关键机制；2）建立内耳干细胞调控和毛细胞再生的研究模型和基因调控工具，实现了成年哺乳动物耳蜗毛细胞的增殖再生；3）联合调控毛细胞发育决定的多个关键基因，实现了成熟耳蜗内毛细胞的功能性再生。上述研究为感音神经性耳聋生物学治疗策略的建立奠定了理论和实验基础。成果发表在Nature Communications（2篇），Cell Reports，Stem Cells等杂志，申请国内发明专利2项，主要研究人员获得国家自然科学基金优青项目（1项）、面上项目（1项）、青年项目（2项）、上海市扬帆计划、超级博士后计划等项目的资助。