ATP-缝隙连接蛋白-P2嘌呤受体功能单位对听觉发育的调控作用

In the course of auditory development, the inner hair cells in the cochlea either fire spontaneous action potentials or fire induced action potentials elicited by ATP release from neighboring supporting cells in the K?lliker organ, thus promoting the maturation of the peripheral auditory organ. However, its regulatory mechanism still remains unclear. We have long been engaged in the research of ATP in the cochlea, and are the first to report the mechanism of ATP release from the marginal cells in the stria vascularis and supporting cells in the K?lliker organ, supporting cell apoptosis in vivo and proliferation in vitro. We found for the first time that P2X7 receptor (P2X7R) expression existed in the cuticular plate of the hair cell, the expression of IP3 receptor was associated with gap junction protein subunit 26 (Cx26), and Cx43 participated in the reorganization and refinement of innervation to the hair cells. These results pushed the understanding of the relationship between ATP and cochlear development forward. Given that the functions of ATP-Cx-P2R are closely related, we assume that it acts as a functional unit in the cochlea, exerting a regulatory role in auditory development. To substantiate this assumption, the present research will detect the coordination of gene transcription of Cx with the enzymes (proteins) relating to ATP synthesis and conversion, P2R, PLC, IP3 receptor, etc., the functional associations among proteins/receptors, and the regulating variation of the functional unit and its role in the reorganization and refinement of innervation to the hair cells. The present research has profound significance for the further elucidation of the developmental mechanism of the peripheral auditory organ.

在听觉发育过程中，耳蜗内毛细胞或发放自发动作电位，或受邻近Kolliker器支持细胞释放的ATP作用发放诱发动作电位，促进外周听器的成熟，但其调控机制不明确。申请人长期从事耳蜗中ATP的研究，首次报道血管纹缘细胞和K?lliker器支持细胞释放ATP的机制，Kolliker器支持细胞在体凋亡、离体增殖，率先发现P2X7嘌呤受体在毛细胞表皮板表达，IP3受体的表达与缝隙连接蛋白26(Cx26)关联，Cx43参与毛细胞神经支配重组。以上结果将ATP与耳蜗发育关系的理解推进了一步。鉴于ATP-Cx-P2R功能紧密关联，我们推测其是一个功能单位，在听觉发育中发挥调控作用。为证实该假设，本项目拟检测Cx与ATP合成/转化有关的酶（蛋白）、P2R、PLC、IP3受体等是否存在基因转录协调性，蛋白/受体功能关联，功能单位的调控变化、在毛细胞神经支配重组中的作用。本研究对进一步阐明听觉发育机制有重要意义。

在听觉发育过程中，耳蜗内毛细胞或发放自发动作电位，或受邻近 Kölliker 器（KO）支持细胞释放的 ATP 作用发放诱发动作电位，促进外周听器的成熟。本项目的主要研究内容包括支持细胞缝隙连接蛋白与 ATP 合成/转化有关的酶(蛋白)、P2 嘌呤受体、PLC、IP3 受体、 RIBEYE 和 otoferlin 等是否存在基因转录的协调性，受体之间功能的关联性，表观遗传学修饰对功能单位的调控、及其在听觉发育中的作用，以及KO退化消失的机制。主要结果及简要意义如下，通过转录组测序发现P1与P7期细胞组分存在差异表达；P1与P14比较显示基因表达差异主要富集在组织器官发育的生物过程方面，提示在P14天，促进耳蜗功能发育的基因表达占主要比例；免疫共沉淀实验提示PLCγ1与P2X2，P2Y4，P2Y7都存在蛋白相互作用,Cx30、Cx43、CX26两两蛋白之间存在相互作用。Cx30、Cx43、CX26与V-ATPase蛋白均存在相互作用。Cx30与ADCY3存在蛋白相互作用，Cx43、CX26与ADCY3之间不存在蛋白相互作用。CX26甲基化修饰蛋白CHD1，可以调控功能单位不同时期蛋白的表达，经耳蜗中阶灌注CHD1-siRNA慢病毒可新生大鼠听力下降；自噬相关标记物均表达于KO支持细胞中，说明KO退化消失的进程中存在着自噬性死亡，并且自噬的发生要早于凋亡。