耳蜗发育早期Kölliker器支持细胞自噬依赖性ATP产生和释放的分子机制研究

三磷酸腺苷（ATP）对于耳蜗Kölliker器的退化及发育早期自发性电活动非常重要，但Kölliker器支持细胞ATP产生和释放的机制仍不清楚。课题组率先发现Kölliker器支持细胞内ATP储存于溶酶体中；出生后早期Kölliker器存在高水平的自噬，而体外抑制Kölliker器自噬水平，能够抑制ATP的产生和释放。我们推测Kölliker器支持细胞存在自噬依赖性、经溶酶体胞吐的ATP释放。其可为支持细胞的凋亡提供ATP作为能量，同时为Kölliker器自发性节律性释放ATP提供来源。本项目拟构建支持细胞自噬相关基因Atg5缺陷小鼠，通过免疫荧光、生物发光法、形态学、分子生物学和钙成像等研究，观察ATP的产生和释放与溶酶体的关系，并观察Kölliker器形态和凋亡改变以及自发性钙信号活动。本项目首次探讨Kölliker器自噬依赖性ATP产生和释放的机制，对深入理解听觉发育机制有重要意义。

Adenosine triphosphate (ATP) is crucial for the degeneration of the Kölliker’s organ and spontaneous electrical activity in the early stage of cochlea development, however, the mechanism underlying ATP production and release from Kölliker’s organ supporting cells remain unclear. We found for the first time that ATP in Kölliker’s organ supporting cells is stored in the lysosomes; there is a high level of autophagy in the Kölliker’s organ in early postnatal period, while the inhibition of autophagy in vitro can suppress the production and release of ATP. We hypothesize that an autophagy-dependent ATP release through lysosomal exocytosis exists in Kölliker’s organ supporting cells by which ATP can be provided as energy for the apoptosis of supporting cells and as a source for the spontaneous rhythmic release of ATP in the Kölliker’s organ. In this project, we are to construct autophagy related genes-ATG5 deficient mice, and observe the relationship between the production and release of ATP and the lysosome, the changes of the morphology and apoptosis level, as well as spontaneous calcium signal activity in the Kölliker’s organ, through a series of experiments including immunofluorescence, bioluminescence, morphology, molecular biology and calcium imaging. This project is the first to explore the mechanism underlying autophagy-dependent ATP production and release in the Kölliker’s organ, which is of great significance to further understand the mechanism of auditory development.