边缘细胞NKCC1的功能性表达与老年性聋的发病机制

NKCC1, KCNQ1 and Na, K-ATPase are three potassium transport proteins present in the marginal cell (MC) membranes. They play critical roles in auditory function in the inner ear by active potassium transport, endochochear potential (EP) formation and maintenance. Presbycusis also known as age-related hearing loss (AHL) is the result of various types of physiological degeneration plus the accumulated effects of noise exposure, medical disorders and their treatment, as well as hereditary susceptibility. Our previous studies have showed that NKCC1 located in the basolateral membrane and KCNQ1 located in the apical membrane of MC are involved in pathogenesis of presbycusis, while α2Na, K-ATPase, that is locaed in the spiral ligament, has little effect on presbycusis.However,the role of NKCC1 in auditory function and in presbycuss remains unclear and needs to elucidate. ..We will utilize various transgenic mice (such as C57BL/J,CBA/J,BAL B/C and knockout mice of KCNQ1+/-, NKCC1+/-, KCNQ1+/-/NKCC1+/-and NKCC1+/-/α1Na, K-ATPase+/-) as experimental models of presbycusis and investigate the role of NKCC1 in the auditory function and in pathogenesis of presbycusis, and the possible inter-relationship of the three potassium ion channels. The model mice will be studies at varied ages in terms of auditory physiology, inner ear morphology (histology and electronic microscopy), protein intracellular localization (immunohistochemistry and confocal microscopy) and protein expression quantification (Western blot and Elisa), and determination of potassium transport protein activity(clamp patch). By combining results from the functional, morphological and cellular-molecular biological study, we may well learn how NKCC1 contributes to auditory function and relationship of NKCC1 deficit/dysfunction and age-related hearing loss to presbycusis pathogenesis. This will help to enrich cochlear potassium ion circulation and hearing mechanism. Also the finding in our experimental study will provide new strategies to prevent and treat of prevention and treatment of the disease.

NKCC1、KCNQ1、Na,K-ATPase三种钾离子转运蛋白对于耳蜗钾离子循环、EP的形成和维持及听觉功能至关重要。老年性聋是多因素作用引起的年龄相关性听力损失。前期的研究发现NKCC1、KCNQ1与老年性聋发病有一定关联,而螺旋韧带的α2Na,K-ATPase对老年性聋影响甚微,为了阐明NKCC1在听觉和老年性聋发病中的作用,我们采用不同基因型小鼠(KCNQ1+/-, NKCC1+/-, KCNQ1+/-/NKCC1+/-, NKCC1+/-/α1Na,K-ATPase,C57BL/6，CBA/J,BAL B/C)作为实验对象建立老年性聋动物模型,利用听觉生理学、内耳形态学、细胞培养、分子生物学和膜片钳等方法研究边缘细胞NKCC1的功能性表达和年龄相关性变化。本课题将从新的视角阐明老年性聋的具体发生机制,为完善以耳蜗钾循环为主的听觉机理提供实验理论依据,为老年性聋的防治提供新的靶点。

老年性聋是多因素作用引起的年龄相关性听力损失。NKCC1、 KCNQ1、 Na,K-ATPase三种钾离子转运蛋白对于耳蜗钾离子循环、 EP的形成和维持及听觉功能至关重要。前期的研究发现NKCC1、KCNQ1与老年性聋发病有一定关联。本课题通过利用ABR、 DPOAE等听觉生理检测技术， EP等电生理技术，以及电镜、免疫荧光、边缘细胞培养方法，对各系小鼠进行了关于NKCC1钾离子转运蛋白的功能研究. 用免疫组织化学、 激光共聚焦显微技术、免疫电镜、 RT-PCR、 Westernblot、 ELISA等方法检测NKCC1离子转运蛋白在血管纹边缘细胞的定位，以及其在不同种系不同鼠龄小鼠血管纹组织中表达差异。结果，KCNQ1杂合子小鼠、 NKCC1杂合子小鼠EP随年龄下降，具有AHL特点，其发病与相应的离子转运蛋白NKCC1表达下降有关；NKCC1+/-KCNQ1+/-双杂合子小鼠具有稳定的听力，其EP在各年龄段相对保持稳定；C57BL/6J小鼠也具有AHL特点，NKCC1主要表达于耳蜗血管纹边缘细胞底侧膜，随年龄增加，表达量也明显下降，其EP也随年龄下降，推测其发病与相应的离子转运蛋白NKCC1表达下降有关。综上，初步阐明了NKCC1离子转运蛋白的在耳蜗中的分布、年龄相关性表达、在EP形成过程中的作用，及在AHL发病过程中的变化。初步明确EP和K+循环在老年性耳聋发生中的内在相互作用机制，明确血管纹边缘细胞NKCC1与老年性耳聋发病的确切机制，有助于筛选潜在的 AHL 致病靶点基因和离子通道，进一步明晰完善耳蜗 K+循环理论，为完全阐明 AHL 发病机制提供新思路。