利用基因敲除小鼠研究ILDR1基因在感音神经性耳聋中的作用及机制

Sensoryneural hearing loss is one of the most common diseases associated with sensory defects. In recent years, more and more new deafness related genes were identified. However, the pathological study of deafness related genes are far from enough. ILDR1 was a newly identified deafness related gene with poor functional characterization. Until now, the molecular mechanism of deafness resulted from ILDR1 dysfunction is still unclear.. In this study, we will explore the mechanism of deafness resulted from ILDR1 dysfunction by using the ILDR1 knockout mouse. Previously, we generated ILDR1 knockout mouse model successfully. Hearing ability tests revealed total deafness in homozygous knockout mice. The structure of the organ of corti was observed using paraffin section combined with HE staining and the morphology of hair cells and supporting cells was observed using cryostat section combined with immunostaining; Hair bundles and organelles in hair cells were observed using scanning electron microscope (SEM) and transmission electron microscope (TEM) respectively. Furthermore, we perform the mass spectrometry analysis of cochlea proteins and SBP-pull-down to find out the ILDR1 interacting proteins. Subsequently, the pathway ILDR1 participated in was confirmed through functional verification.. Finally, through in vivo and in vitro study, we hoped to fully uncover the function and the pivotal roles of ILDR1 in hearing formation which would provide theoretical foundation for the discovery of new mechanism for deafness.

感音神经性耳聋是人类最常见的感官或功能缺陷性疾病。目前，越来越多的致聋新基因被发现。但是，致聋基因致病机制的研究仍不够深入。ILDR1是近年来新发现的致聋基因。迄今为止，该基因在听力形成中的作用及导致耳聋的分子机制尚不清楚。. 本课题拟利用基因敲除小鼠模型对ILDR1的致聋机制进行深入研究。前期已成功构建ILDR1敲除小鼠模型，听觉检测发现ILDR1纯合敲除小鼠表现为全聋。后期拟利用石蜡切片结合HE染色、冰冻切片结合免疫荧光观察耳蜗Corti器和内外毛细胞、支持细胞的形态；利用扫描电镜和透射电镜观察毛细胞纤毛束和毛细胞内细胞器的结构变化。最后进一步通过耳蜗蛋白质谱分析和SBP-pull-down找出ILDR1相互作用蛋白并进行相应功能验证，从而确定ILDR1所参与的信号通路。最终通过体内、体外研究，全面揭示ILDR1的功能及其在听觉形成中的重要作用，为新致聋机制的发现提供理论依据

ILDR1是一个具有免疫球蛋白样结构的受体膜蛋白，最初在淋巴瘤中被克隆出来。ILDR1突变会导致耳聋，但其致聋机制一直未知。为研究ILDR1的致聋机制，我们构建了ILDR1敲除小鼠。ILDR1表达于小鼠耳蜗corti器的内毛细胞和外毛细胞。ILDR1敲除的纯合小鼠在第15天表现为轻度耳聋，内耳支持细胞和内毛细胞正常，但外毛细胞开始部分退化。21天时，纯合小鼠表现为重度耳聋，外毛细胞继续退化，corti器开始塌陷。28天时，纯合小鼠表现为全聋，外毛细胞全部消失，corti器完全塌陷。ILDR1敲除小鼠耳蜗中tricellulin蛋白定位异常。为进一步研究致聋机制，我们对21天野生和纯合小鼠耳蜗蛋白进行质谱分析，发现有708个蛋白上调，114个蛋白下调，基因聚类分析发现，大部分差异蛋白参与细胞链接，蛋白转运，细胞死亡，膜组装和细胞稳态等生理功能。综上，我们的研究结果显示，ILDR1对耳蜗外毛细胞的生存至关重要，这对ILDR1导致耳聋的机制进行了深入阐明。