Cav1.3钙离子通道的年龄相关性表达与老年性耳聋的研究

Calcium ions (Ca2+) regulate numerous and diverse cellular activities of cochlea hair cells, such as mechanical transduction, adaptation, tuning. Ca2+ homeostasis in the cochlea is fundament of fully functioning of the auditory system. As the primarily calcium channels in the inner ear, Cav1.3 calcium channels control calcium influx and membrane depolarization of hair cells and promote neurotransmitter release and nerve impulse conduction. Also, Cav1.3 calcium channels are essential for cochlear development, Ca2+ homeostasis and auditory functioning. Currently, the research on Cav1.3 calcium channel mainly focuses on the hair cells. However, the underlying mechanism of Cav1.3 calcium channels in the inner ear development, calcium ion transport and presbycusis remains unclear. Utilizing knockout mice, such as Cav1.3+/+,Cav1.3+/-,Cav1.3-/-,C57BL/6J,we will obtain experimental models that present various hearing characteristics. Auditory physiology, inner ear morphology, immunohistochemistry, molecular biology, and calcium fluorescence probe techniques and methods will be applied to detect the calcium ion concentration gradient in inner ear tissue and the aged related expression of Cav1.3 calcium channels. Thereby our study results will explore the role of Cav1.3 calcium channels in the development of cochlea, Ca2+ homeostasis and the pathogenesis of Presbycusis. This study will enrich and improve the inner ear calcium ion transport model, and therefore provide new ideas and strategies for the prevention and treatment of Presbycusis.

钙离子参与毛细胞的机械转导、适应、调谐等多种细胞活动，耳蜗内钙离子稳态是听觉功能正常发挥的基础。Cav1.3是内耳主要的钙离子通道，主要功能是控制毛细胞钙离子内流和细胞膜的去极化，促进神经递质释放和神经冲动的传导，其在听觉功能中发挥了重要的作用。目前对于Cav1.3的研究主要集中在毛细胞，而Cav1.3在内耳发育、钙离子转运及老年性耳聋中的作用尚不明晰。本研究拟采用Cav1.3+/+，Cav1.3+/-，Cav1.3-/-及C57BL/6J小鼠作为实验动物模型，运用听觉生理学、内耳形态学、免疫组织化学、分子生物学、钙离子荧光探针等技术和方法检测内耳组织钙离子浓度梯度及Cav1.3在不同基因型小鼠内耳中的年龄相关性表达，探讨Cav1.3在内耳发育、耳蜗钙离子稳态及老年性耳聋中的作用机理，本研究将丰富和完善内耳钙离子转运模型，并为老年性耳聋的防治提供新的思路和策略。

钙离子参与毛细胞的机械转导、适应、调谐等多种细胞活动，耳蜗内钙离子稳态是听觉功能正常发挥的基础。Cav1.3是内耳主要的钙离子通道，主要功能是控制毛细胞钙离子内流和细胞膜的去极化，促进神经递质释放和神经冲动的传导，其在听觉功能中发挥了重要的作用。本课题研究初步揭示Cav1.3钙离子通道可能与老年性耳聋的发病有一定的关联，我们利用SD大鼠及C57BL/6J小鼠作为动物模型，运用免疫荧光、QRT-PCR、蛋白印迹、边缘细胞原代培养、流式细胞学等方法，研究了Cav1.3钙离子通道在SD大鼠及C57BL/6J小鼠不同发育阶段内耳及听觉中枢的表达。结果显示，Cav1.3钙离子通道在原代培养的新生大鼠耳蜗血管纹边缘细胞上表达阳性；Cav1.3钙离子通道在出生后0天、6天、14天、21天大鼠的耳蜗外侧壁和基底膜的表达逐渐增多；成年大鼠耳蜗中Cav1.3钙离子通道主要分布在大鼠耳蜗内外毛细胞的侧膜和顶膜、血管纹细胞，螺旋神经板缘细胞、螺旋韧带、螺旋神经节、螺旋凸。其中耳蜗组织中基底膜上最多，螺旋神经节次之，耳蜗侧壁血管纹和前两者相比其表达的量相对较少；Cav1.3钙离子通道在小鼠听皮层、海马、下丘神经核、耳蜗核中均有表达，4~16周小鼠随年龄增长听皮层中Cav1.3的表达逐渐增强，16周以后Cav1.3表达呈现下降趋势。下丘神经核中Cav1.3表达水平在4~8周呈现上升趋势，16周以后Cav1.3的表达下降。海马中Cav1.3表达量在第4周以后出现下降并保持在较低水平，耳蜗核中Cav1.3表达水平随年龄变化没有明显改变。综上所述，本研究初步揭示了Cav1.3钙离子通道可能参与了内耳的发育与听觉功能的形成；Cav1.3钙离子通道在耳蜗、听觉通路及听觉中枢不同组织间的选择性及差异性表达与听觉功能密切相关；为老年性耳聋的防治提供新的思路和策略，为进一步揭示钙离子通道在听觉通路中作用机制提供了理论基础。