微丝骨架在耳蜗毛细胞表皮板和静纤毛发育过程中的功能分析

The sound is transmitted to the central auditory system through the transformation of the cochlear hair cells into electrical signals. The stereocilia at the tip of the hair cells are important in the process of the animal's formation of the sensation of hearing. The stereocilia contains a tightly-associated, parallel filamentous actin (F-actin) bundle organized in a paracrystalline array. The stereocilium tapers at its base, and a few actin filaments continue as a densely-packed rootlet, which inserts into the F-actin matrix of the cuticular plate that underlies all stereocilia. Actin is an important component of the composition of the stereocilia and the cuticular plates, regulating the development and function of the stereocilia and the cuticular plates. Due to the limitation of microscopic technology, the formation process of the stereocilia and the cuticular plates is still not clear so far. A series of questions such as how to accumulate actin in the cuticular plates, how the F-actin is assembled in the stereocilia, and what kind of high-order structure the F-actin forms, are not well understood. This project will use super-resolution fluorescence microscopy to observe the cuticular plates and the stereocilia of hair cells at different developmental stages, and combine the immunolabeling technique to observe the F-actin skeleton and actin binding proteins at different developmental stages. Analyze the period positioning mode and organizational process of F-actin. This project has important research significance which will advance our understanding of the developmental process of the cuticular plates and the stereocilia of cochlear hair cells, and provide a basis for further study on the developmental regulation of the cuticular plates and the stereocilia.

外界的声音通过耳蜗毛细胞转化为电信号传入听觉中枢神经系统产生听觉，毛细胞顶端的纤毛在动物形成听觉的过程中及其重要。静纤毛中含有紧密的平行排列的微丝束，部分微丝在其底部形成致密根部插入到富含肌动蛋白的表皮板中。微丝是组成静纤毛和表皮板的重要成分，调控静纤毛和表皮板的发育和功能维持。由于显微技术的限制，目前为止对纤毛和表皮板的形成过程还不是太清楚，对肌动蛋白在表皮板中如何聚集、微丝在静纤毛中如何组装、微丝形成何种高级结构、由哪些蛋白调控等一系列问题也不甚了解。本项目将利用超分辨荧光显微镜对不同发育时期时期的毛细胞的表皮板和静纤毛进行观察，结合免疫标记技术，对不同发育时期的微丝骨架及微丝骨架结合蛋白进行观察，分析其在不同时期定位模式及组织过程。本项目的开展将推进我们对耳蜗毛细胞表皮板和静纤毛的发育过程的理解，并为进一步研究表皮板和静纤毛的发育调控提供了基础，具有重要的研究意义。

本项目按照计划对毛细胞进行不同发育时期的微丝染色进行超分辨观察；对多种微丝结合蛋白、微丝相关蛋白和纤毛发育相关蛋白进行染色观察；利用knock in技术引入报告基因，目前已成功利用AAV病毒将报告基因转入基因组，并进行超分辨显微观察，为今后对其进行活体观察打下基础；进行体外活体耳蜗培养实验；利用质谱技术对耳蜗蛋白进行分析，寻找更多耳蜗中的细胞骨架相关蛋白；为今后对耳蜗毛细胞表皮板和纤毛发育的进一步研究打下基础。