Wnt信号通路在math1诱导的毛细胞再生后听觉感觉上皮平面细胞极性重塑中的作用

Lose of mechanosensory hair cell leads to irreversible hearing loss. Ectopic new hair cell has been shown to be produced from non-sensory cells or stem cells by gene or growth factors inducement. However, it is needed the precise mosaic structure with new hair cell and relative supporting cell and the remodeling of planar cell polarity(PCP), which means the consistent orientation of adjacent hair cells and the convergent extension movement during sensory epithelium formation to restore the complex structure of hearing sensory epithelium. As little is known about these two aspects, a hair cell regeneration model by math1 overexpression was established in our experiment, in which new hair cell-supporting cell combination structures has been observed and PCP phenomenon has been well repeated indicated with cell orientation formation in new hair cells and convergent-like movement in new sensory epithelium. Our researches including the following objects: ①To observe the details of new hair cell regeneration, including morphological changing, kinocilia and stereocilium formation, stererocilium orientation process by confocal microscope and scanning electron microscope;②To explore the mechanism of new supporting cell formation by membrane protein;③To inquire the role wnt signal pathway in PCP by using ligands or inhibitors in culture system and by polarity molecular labeling;④To compare the consistency and difference in hair cell-supporting cell structure formation and planar cell polarity commence between hair cell regeneration and development by math1-EGFP mice(ready to use). The significant of our experiment is to expound the mechanism of new hair cell-supporting cell structure and PCP formation, and to provide a further theoretical basis for hearing restore from single cell type production to the whole sensory epithelium remodeling.

听觉毛细胞损失导致不可逆性耳聋。目前可通过基因诱导等方法获得新生毛细胞，然而新生毛细胞能否修复听觉感觉上皮还需支持细胞的精确支撑及平面细胞极性(PCP)的恢复，目前尚无针对性报道。我们已利用病毒感染过表达math1诱导毛细胞再生，并成功观察到新生毛细胞-支持细胞组合的形成及PCP事件的发生（新毛细胞极性产生及定向运动），拟在此基础上研究其发生和分子机制，具体包括：①利用形态学方法研究毛细胞再生过程，动、静纤毛束发生和定向规律；②研究math1表达与新毛细胞和新支持细胞分化及组合的关系；③利用配体或抑制剂调控wnt信号通路（调控PCP），并通过极性分子特异性标记，研究PCP事件的分子机制；④利用math1-EGFP转基因小鼠(已备)对比上述再生过程与发育过程分子机制的异同。通过上述研究，力图阐明新生毛细胞与支持细胞组合结构形成及PCP重塑的分子机制，为从毛细胞新生到听觉上皮恢复提供理论基础。

听觉毛细胞损失导致不可逆性耳聋。目前可通过基因诱导等方法获得新生毛细胞，然而新生毛细胞能否修复听觉感觉上皮还需支持细胞的精确支撑及平面细胞极性(PCP)的恢复，目前尚无针对性报道。本课题重要结果①利用病毒感染过表达math1诱导毛细胞再生，发现不同来源的新生毛细胞其转化过程并不相同（部分结果发表，Neuroscience letters, 549（2013）7-11）。②新生静纤毛可能由微绒毛的延长产生，纤毛侧连接伴随其直立过程，可能对其成束起一定作用；③发现新生毛细胞-支持细胞组合的形成，其中毛细胞和支持细胞样细胞分别表达myosin7a以及GFAP的特征性蛋白；且新的毛细胞和支持细胞在膜片钳测试下分别产生了不同的电生理特性。④非感觉上皮受感染后的汇聚运动，并呈现向底圈趋化的特性，这与发育过程相似。⑤新感觉上皮的汇聚运动，依赖于细胞链接的存在。影响细胞链接时，汇聚运动收到干扰，上皮宽度差别具有统计学意义（部分结果已发表，Brain Research，1615（2015）22-30）。通过上述研究，第一次提出新生毛细胞与支持细胞功能组合结构同时新生的概念，并第一次在体外组织实现平面细胞极性并探讨其机制，为从单个毛细胞新生到听觉上皮整体功能再生提供理论依据。