血影蛋白βV维持耳蜗毛细胞形态和功能稳定的分子机制

The remarkable hearing sensitivity and frequency selectivity of mammals’ auditory system depend on the singular structures of cochlear hair cells, including their molecular architecture. The family of spectrin, one of the molecular structures of hair cells, has attracted more attention from otology and neuroscience researchers. Mutations in spectrins associate with deafness, epilepsy and progressive encephalopathy. In our present study, we generated Sptan1f/f mice for deletion of spectrin αII of inner hair cells (IHCs) and outer hair cells (OHCs), and analyzed the morphological and functional changes of spectrin αII deficient mice. In spectrin αII knockout mice, both IHCs and OHCs are intact at birth, IHCs showed few missing until 1 month, but OHCs progressively died and about 75% OHCs were lost at 1 month. Spectrin αII is distributed equally in normal IHCs and OHCs, and low or no expression in spectrin αII deficient IHCs and OHCs. One hypothesis for the discrepancy between IHCs and OHCs is that OHCs have more complicated proteins interactive networks, including spectrin βV. In addition, the spectrin βV has the same time-space distribution patterns as the lateral plasma membrane protein prestin in OHCs, and a role of spectrin βV in OHCs’ electromotility has been hypothesized. To confirm these hypotheses, this project will explore the role of spectrin βV in cochlear hair cells at the molecular level, cell level and animal level, using knockout mouse model, RNA interference and proteins interaction methods, together with auditory electrophysiological and patch clamp techniques. This will help promote our understanding of the three dimensional molecular structures of hair cells and cochlear physiology, and provide cues for the new therapeutic methods for sensorineural deafness in the future.

听觉的高度敏感性和频率选择性与耳蜗毛细胞的分子构成密切相关，其中血影蛋白家族日益引起研究者关注。本课题组前期研究发现Sptan1敲除小鼠耳蜗内、外毛细胞血影蛋白αⅡ表达均减少但内毛细胞形态学基本正常，外毛细胞进行性死亡。我们推测内、外毛细胞存活的差异可能与外毛细胞膜内侧复杂的蛋白网络有关，即血影蛋白βV与αⅡ及Prestin等相互作用。此外，既往研究表明血影蛋白βV和Prestin在外毛细胞的表达存在时间和空间的一致性，推测血影蛋白βV可能参与外毛细胞声诱发的电能动性。为证实上述两种假说，本项目拟在分子水平、细胞水平和整体动物水平，通过蛋白质相互作用、RNA干扰、基因敲除小鼠模型，同时结合听觉电生理和膜片钳等技术，研究血影蛋白βV在毛细胞特别是外毛细胞中的作用。这将有助于推动我们对毛细胞三维分子结构和耳蜗生理功能的认识，也可能为未来探索感音神经性聋全新的治疗方法提供线索。

听觉的高度敏感性和频率选择性与耳蜗毛细胞的分子构成密切相关，其中血影蛋白家族日益引起研究者关注，我们前期研究发现αⅡ血影蛋白对于毛细胞静纤毛结构稳定和功能维持具有重要作用。在本项目中，我们构建Sptbn5 毛细胞条件敲除小鼠构建，揭示Sptbn5 mRNA在内耳、肺、气管与睾丸等器官有较高表达，βV血影蛋白在小鼠耳蜗毛细胞、螺旋神经节有表达，在前庭毛细胞静纤毛根部周围有表达。βⅤ血影蛋白敲除小鼠基底膜无毛细胞缺失，静纤毛形态未见明显异常。通过基底膜毛细胞对FM1-43摄取研究毛细胞的功能状态，结果发现敲除鼠和对照鼠耳蜗毛细胞功能无明显区别。与对照组小鼠相比，血影蛋白βⅤ敲除小鼠ABR阈值明显升高。在HEI-OC1细胞和动物水平，通过RNA干扰、Western blot、免疫荧光等研究Sptan1敲除后通过影响Integrin β1，FAK、Paxillin、Vinculin、SPTBN1等蛋白的表达，提示血影蛋白通过粘着斑信号通路发挥作用。本项目通过多种技术揭示βV血影蛋白与听功能的维持密切相关。这将有助于推动对毛细胞三维分子结构和耳蜗生理功能的认识，也可能为未来探索感音神经性聋全新的治疗方法提供线索。

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