【作者】 王晓颖；

【导师】 徐志刚；

【作者基本信息】 山东大学 ， 发育生物学（专业学位）， 2022， 硕士

【摘要】 生长于内耳毛细胞顶端、呈阶梯状排列的静纤毛结构是动物感知声音和运动信息的重要结构基础,其顶端的MET通道负责将机械信号转换为电信号,以此向神经中枢传递声音和运动信息。静纤毛发育以及MET通道的组成和功能调控机制是内耳研究的热点问题。CIB2与CIB3同属CIB蛋白家族,它们的功能结构高度相似,被认为是MET通道的辅助亚基。CIB2定位于静纤毛顶端,能够参与听毛细胞纤毛发育及MET功能行使。其缺失会导致听毛细胞静纤毛形态异常、MET功能受损,小鼠听力严重丧失,但其前庭功能并无明显异常。CIB2与CIB3在小鼠前庭中都有表达,但对CIB2及CIB3蛋白在前庭毛细胞中的功能还缺乏系统和深入的讨论,并且它们之间是否存在功能重叠也是未知的。本研究通过对Cib2和Cib3单基因及双基因敲除小鼠前庭形态与功能分析比较,较为系统地探讨了 CIB2和CIB3在前庭中的功能。首先利用毛细胞电转染的方式,我们发现CIB2和CIB3蛋白均定位于前庭毛细胞静纤毛的顶端,但原位杂交结果显示,相较于Cib3在前庭纹外区中的特异性表达,Cib2在微纹区和纹外区都有表达,且在微纹区的表达略高于纹外区。进一步分析敲除小鼠前庭毛细胞静纤毛的发育及前庭功能,我们发现CIB2缺失导致小鼠前庭微纹区静纤毛较低排变细、总排数减少,并在平衡木测验中表现出异常。CIB3缺失对小鼠前庭静纤毛影响较弱,但随着年龄增长,小鼠平衡能力也出现异常。当CIB2与CIB3共同缺失时,前庭静纤毛发育异常及功能紊乱加剧。扫描电镜结果显示,Cib2-/-;Cib3-/-小鼠微纹区和纹外区的静纤毛均出现明显异常,静纤毛排数显著减少。MYO7A和phalloidin染色表明Cib2-/-;Cib3-/-小鼠前庭毛细胞和静纤毛密度有所降低。行为学检测发现,Cib2-/-;Cib3-/-小鼠出现明显的转圈行为,在转棒上停留时间显著缩短,无法正常游泳。综上所述,我们发现CIB2与CIB3共同参与前庭静纤毛的发育和维持,调节前庭的功能,并且二者的功能有一定的重叠,CIB2对前庭微纹区静纤毛的发育和维持以及毛细胞功能行使不可或缺,而纹外区则主要由CIB3发挥作用。本研究结合敲除小鼠前庭表型及功能分析,对同源蛋白CIB2及CIB3在前庭中的功能进行了较为系统的研究,为深入了解前庭不同区域毛细胞静纤毛发育及功能调控的分子机制提供了可靠的研究数据。更多还原

【Abstract】 The stair-like structure of stereocilia growing on the top of inner ear hair cells is important for animals to perceive sound and motion information.The MET channel at the top of stereocilia is responsible for converting mechanical signals into electrical signals,so as to transmit sound and motion information to nerve center.The development of stereocilia and the regulation mechanism of MET channel composition and function are hot topics in inner ear research field.CIB3 and CIB2 belong to the same family of CIB proteins,and their structures are highly similar to each other.Recently,they are proved to be auxiliary subunits of the MET channel of hair cells.CIB2 located at the tip of stereocilia has been shown to be involved in the development of stereocilia of auditory hair cells and the function of MET.Loss of CIB2 leads to impaired MET function,abnormal cochlear stereocilia morphology and severe hearing loss in mice.However,there are no obvious abnormal vestibular function.Both Cib2 and Cib3 are expressed in the vestibule,while the functions of CIB2 and CIB3 proteins in vestibular hair cells are lack of systematic discussion,and whether there is functional overlap between them or not is unknown.In this study,we systematically explore the expression and function of CIB2 and CIB3 in the vestibule by comparing the morphology and function of the vestibule in Cib2 and Cib3 single-gene and double-gene knockout mice.By injectoporation,we found that both CIB2 and CIB3 proteins can localize at the tip of the vestibular hair cell stereocilia,but the results of in situ hybridization show that compared with the specific expression of Cib3 transcripts in the vestibular extrastriola,Cib2 transcripts are expressed in both striola and extrastriola,and the expression in striola is slightly higher than that in extrastriola.Further studies on the vestibule of knockout mice reveal that CIB2 deletion leads to thinning stereocilia in lower rows and a decrease in the number of rows in vestibular striola.Moreover,Cib2-/-.mice have difficult in traveling the balance beam.Loss of CIB3 also causes a mild defect on the vestibular stereocilia in mice,and Cib3-/-mice exhibit abnormal balance with age.The co-deletion of CIB2 and CIB3 aggravates the abnormal development and dysfunction of vestibular stereocilia.Scanning electron microscopy shows the abnormal morphology of stereocilia of vestibular hair cell in Cib2-/-;Cib3-/-mice.MYO7A and phalloidin staining suggest that the density of vestibular hair cells and stereocilia decreases in Cib2-/-;Cib3-/-mice.Behavioral tests show that the mice have obvious circling behavior and can not swim normally.Their residence time on the rotarod also decreases significantly.In summary,we suggest that CIB2 and CIB3 are jointly involved in the development and maintenance of vestibular stereocilia.They regulate the function of the vestibule together,and the functional areas of the two are complementary to a certain extent.In the striola,CIB2 undertakes the function alone,while CIB3 plays a major role in the extrastriola.In this study,combined with the vestibular phenotype and functional analysis of knockout mice,we systematically discuss the function of homologous proteins CIB2 and CIB3 in the vestibule.We also provide reliable research data for a deeper understanding of the molecular mechanisms that regulate the development and function of stereocilia of hair cells in different regions of vestibule.更多还原