噪声引起的耳蜗微血管周围巨噬细胞损伤及其对血-迷路屏障的影响

The capillary network of the cochlear stria vascular winds through the gap of intermediat cells, surrounded by foot processes of marginal cells and basal cells, and builds the blood-labyrinth barrier(BLB).The BLB is critical in maintaining endocochlear potential, ion transport, and fluid balance in the inner ear. Disruption of the BLB has long been considered a major etiologic factor in a variety of hearing disorders. Despite the importance of the BLB, the mechanisms that control BLB permeability remain largely unknown. Recent studies have found that there were a large number of perivascular resident macrophage (PVMs) in the BLB. The PVMs are a hybrid cell type which also show some characteristics of melanocytes. They are in close contact with microvascular endothelial cells(ECs) through cytoplastic processes and possibly participate in the regulation of BLB permeability. In this study, we will focus on the PVMs in the blood-labyrinth barrier by in vivo and in vitro models,and explore PVMs development by using homozygous mutant(Mitf-/-) Rongchang swine.The proposed study will invovle a broad range of advanced techniques, including electrophysiology，measurement of vascular permeability,Real-time PCR,siRNA transfection, and functinal in vitro assessment of endothelial junctions.The aim of this research program is to explore the biological characteristics of PVMs and its role in controlling BLB function.We will also observe cochlear PVMs response to acoustic trauma and accompanying changes in BLB and endochlear potential, so as to provide theoretical basis for the prevention and control of noise damage.

耳蜗外侧壁血管纹的毛细血管网穿行于中间细胞的间隙，被边缘细胞和基底细胞的突起所包围，形成血-迷路屏障（BLB），BLB对维持耳蜗内电位、离子转运以及内耳液体平衡具有重要作用， BLB被破坏会导致各种听力障碍，但控制BLB通透性的机制仍不清楚。晚近的研究发现耳蜗血管纹的微血管周围巨噬细胞兼具黑色素细胞特征，通过突起与微血管内皮细胞（ECs）密切接触，可能参与BLB通透性的调控。本课题以耳蜗外侧壁BLB上的微血管周围巨噬细胞（PVMs）为切入点，拟通过在体器官研究和离体细胞培养两个方面，利用Mitf基因突变型荣昌猪来研究PVMs的发育，利用电生理、血管通透性测量、RT-PCR、 siRNA转染以及体外评估内皮连接的功能等技术，探讨耳蜗PVMs的细胞生物学特性及其对血-迷路屏障的调控，以及噪声引起的耳蜗PVMs损伤及其对血-迷路屏障、耳蜗内电位（EP）的影响，从而为噪声损伤的防治提供理论依据。

噪声对耳蜗的损伤是多因素的,包括机械损伤耳蜗内重要结构、耳蜗微血管损伤导致血流及供氧减少、代谢紊乱诱发细胞凋亡及坏死等。其中噪声引起耳蜗毛细胞内缺氧和能量代谢障碍，外毛细胞死亡可能是噪声性听力损伤的重要原因。.采用120dB(A) 白噪声（4小时/天，连续暴露两天）建立噪声性耳聋动物模型（包括豚鼠、小鼠及小型猪），利用免疫组织化学方法、免疫荧光技术，表面制备法耳蜗铺片和激光扫描共聚焦显微镜成像技术清晰观察到血管纹、螺旋韧带处微血管形态；对正常及噪声损伤后耳蜗外侧壁标本进行分层扫描，发现位于血管纹血管周围巨噬细胞（PVM/Ms）的形态发生改变且分布率降低。.噪声损伤引起耳蜗血管纹基质金属蛋白酶代谢紊乱所致的血迷路屏障通透性的改变。利用免疫组织化学、免疫荧光和激光扫描共聚焦显微镜成像、western blot等技术，发现噪声暴露引起耳蜗血管纹基质金属蛋白酶（MMP-2、MMP-9）的表达上调，噪声暴露后血管纹上紧密连接蛋白ZO-1结构变得松散、细胞间出现破口，血管纹毛细血管通透性增加，发现MMPs抑制剂对内耳噪声性损伤具有一定的保护作用，阐释了MMPs及细胞间连接蛋白在噪声损伤中的变化及其在噪声性血-迷路屏障损伤中的可能机制。.在成功构建小型猪稳态噪声性耳聋模型的基础上，利用蛋白质组学iTRAQ技术对噪声损伤后小型猪耳蜗内差异表达蛋白进行筛选，结果噪声暴露后较正常组具有显著差异表达的蛋白质共227个，噪声后1天和7天组显著差异蛋白有162个，利用生物信息学分析方法GO分析、KEGG pathway，免疫荧光染色、western blot等技术，发现噪声损伤急性期内耳的免疫及炎症通路被激活，认为耳蜗内炎症/免疫反应的过度激活可能是噪声性耳聋发生发展的重要机制。.本课题初步阐明噪声损伤引起耳蜗血管纹PVM/Ms形态及功能变化进而引起微循环的改变，内耳供血及供氧下降、氧化应激产物过度堆积及免疫/炎症反应的过度激活导致毛细胞死亡可能是噪声性听力损伤的主要原因。但噪声损伤的分子机制有待深入研究，对噪声性听力损失的有效防治方法需要进一步探索。