内耳支持细胞通过Wnt/β-catenin信号通路调控羊水干细胞向听觉神经元定向分化

Our country has had more than 27 million patients with hearing impairment.Intact spiral ganglion neurons are required for cochlear implantation or conventional hearing amplification as an intervention for sensorineural hearing loss. Treatment strategies to replace the loss of spiral ganglion neurons are needed. Inner ear stem cells derived feeder layer were first proved to successfully promote directional differentiation of amniotic fluid stem cells into auditory-like neurons in a three dimensions culture model, which could be resources for spiral.ganglion regeneration for neural deafness, in our previous study. However, the detailed mechanism and modulation of there results remain unclear. We propose that inner ear stem cells derived feeder layer might trigger Wnt signaling receptors and target, to active certain relevant genetic expressions, to promote neural differentiation. Wnt signaling may play an essential role in triggering and regulating this neurogenesis. Thus, this project aims to investigate the spatial and temporal expression patterns of major proteins and signals of Wnt/β-catenin pathway, such as Wnt, Frizzled, Dsh, β-catenin, during inner ear stem cells differentiation. We further correlate these characteristics with auditory neural amount and functionality. Different conditions were performed including with/without inner ear supporting cells, over-expression or inhibition of wnt signals, to explore the thorough mechanism of differentiation of amniotic fluid.stem cells into auditory-like neurons induced by inner ear stem cells derived feeder layer. The results might contribute to efforts to translate cell-based strategies to the clinic and has important theoretical significance and potential clinical value for inner ear stem cells transplantation.

我国听力障碍患者已经超过2700万，而针对螺旋神经元损伤引起的耳聋，仍没有确切的办法。我们前期研究首次发现：将羊水干细胞接种于内耳支持细胞饲养层表面，形成支持接触连接的三维培养，在不添加外源性神经元诱导因子时，羊水干细胞在体外能定向分化为听觉神经元样细胞，有望解决神经性耳聋问题，然而内耳支持细胞对羊水干细胞的作用及调控机制尚不清楚。我们推测内耳支持细胞引发了Wnt/β-catenin信号通路，参与调控羊水干细胞向听觉神经元定向分化。本研究开展如下试验作进一步验证：在无内耳支持细胞作用下，羊水干细胞分化是否具有Wnt信号参与；在有内耳支持细胞作用下，是否具有Wnt信号参与，活化及抑制Wnt信号通路，羊水干细胞分化过程中Wnt信号主要相关蛋白及信号因子的表达差异及与听觉神经元分化的数量与功能之间的联系，探讨羊水干细胞定向分化的深入机理，为今后羊水干细胞耳蜗内移植治疗神经性耳聋提供科学依据。

项目背景：我们前期研究发现，羊水干细胞与内耳支持细胞直接接触能定向分化为听觉神经元样细胞，有望解决神经性耳聋问题；Wnt信号途径参与调控羊水干细胞的定向分化，推测内耳支持细胞饲养层诱导了Wnt信号通路并促进神经元的分化。本研究证实羊水干细胞分化过程中Wnt/β-catenin信号通路中，GSK3β、Wnt等主要相关蛋白及信号因子的时空表达差异及与听觉神经元分化的数量与功能之间的联系，证实了羊水干细胞向听觉神经元样细胞分化的机理，为今后羊水干细胞耳蜗内移植治疗神经性耳聋提供理论先导与科学依据。.主要研究内容：明确小鼠内耳支持细胞促进羊水干细胞定向分化为神经元的可能调控机制，Wnt/β-catenin信号通路中相关蛋白的表达。.重要结果：羊水干细胞接种密度以及内耳支持细胞来源差异均可能参与调控羊水干细胞向神经元的定向分化。其内在机制可能与Wnt信号、P27kip1表达量等相关。适当增加羊水干细胞的接种比例、采用前庭组织来源的内耳支持细胞作为饲养层、有效调控Wnt信号、过表达P27kip1基因等，均可能有助于促进羊水干细胞向神经元分化，提高效率。.关键数据：羊水干细胞有向神经元定向分化的潜能，无论是与小鼠基底膜还是前庭来源的内耳支持细胞直接接触共培养，都能促使羊水干细胞定向分化为功能性神经元；但基底膜与前庭组织对羊水干细胞定向分化的效果有明显差异，基底膜来源的分化效率较低，仅有37%左右，而前庭来源的分化效率达52%左右；前庭分化得到的神经突起无论从数量还是长度方面比较，都优于基底膜分化。前庭来源内耳支持细胞P27kip1的表达量高于基底膜，在与羊水干细胞直接接触共培养中，前庭分化得到的神经突起在数量和长度方面均优于基底膜，P27kip1的表达量与神经突起的数量与长度呈正相关，说明内耳支持细胞饲养层表达P27kip1增多，可能导致更多的羊水干细胞停止有丝分裂，失去干性；又由于本身具有向神经定向分化的特殊性，最终导致更多的功能性神经元产生。.科学意义: 探寻小鼠内耳支持细胞对羊水干细胞定向分化的机制，有助于深入神经元分化、再生和保护的相关研究，并对耳科基础研究向临床运用转化具有重要的前期指导意义。