耳蜗血管纹微血管再生及其调控机制研究

The blood of cochlea is provided by the spiral ligament and the capillary network of stria vascularis, which is critical for maintaining exchanges between cochlear vessels and intercellular fluid and endocochlear potential required for normal hearing function. Dysfunction of the stria is considered to be one of the etiologies in a number of hearing disorders, including noise-induced hearing loss, age-related hearing loss and sudden deafness. Our research have found that cochlear endothelial cells, pericytes and perivascular resident macrophage are involved in the regulation of cochlear microcirculation. In this study, we will focus on the microvascular angiogenesis of stria vascularis by newly developed culture-based 3D matrigel models. The aim of this research program is to explore the effects of vascular endothelia growth factor (VEGF) on microvascular angiogenesis of stria vascularis. The morphological characteristics of angiogenesis are identified by tissue culture, immunohistochemistry and confocal laser scanning microscope. We will also select new proteins which contribute to microvascular angiogenesis of stria vascularis by isobaric tags for relative and absolute quantitation (iTRAQ). The new selected proteins are applied to culture-based 3D matrigel models to explore the related signal pathway about angiogenesis and regulatory mechanism to provide potential therapy for ischemia-induced hearing dysfunction.

耳蜗血管纹的微血管对于调控耳蜗血流和细胞间液的物质交换、耳蜗电位维持以保障听功能至关重要。噪声性聋、老年性聋、突发性耳聋等感音性聋与血管纹微血管病变导致的耳蜗血流障碍有关。课题组前期的研究发现耳蜗微血管内皮细胞、周细胞和血管周围巨噬细胞参与耳蜗血流的调控，而噪声可引起耳蜗微血管的损伤，本项目以血管纹微血管的再生为切入点，构建离体耳蜗血管纹微血管再生培养模型；观察血管内皮生长因子（VEGF/VEGFR）信号通路对血管再生的影响；利用组织培养、免疫组化和激光共聚焦显微镜技术鉴定再生血管的形态特征；利用同位素标记相对和绝对定量技术（iTRAQ）及生物信息分析技术，遴选血管纹内促进和抑制血管再生的新的蛋白因子，将其应用于微血管再生模型，研究耳蜗血管纹微血管再生的影响因素及调控机制，实现可控的耳蜗血管纹微血管再生，为内耳缺血引起的耳蜗功能障碍提供可能的治疗方法。

耳蜗血管纹的微血管对于调控耳蜗血流和细胞间液的物质交换、耳蜗电位维持以保障听功能至关重要。多种感音性聋与血管纹微血管病变导致的耳蜗血流障碍和内耳炎症反应损伤有关。.本研究通过蛋白组学及生物信息学技术、免疫组织化学方法、免疫荧光技术，蛋白免疫印迹等方法研究正常和病理情况下耳蜗，尤其是血管纹微血管的形态及蛋白的表达情况。.蛋白组学技术检测及生物信息学分析发现噪声暴露后小鼠血管纹K+循环障碍、连接蛋白破坏、细胞凋亡、氧化应激反应及蛋白磷酸化是造成血管纹损伤的主要机制；而衰老小鼠的血管纹差异蛋白主要富集在细胞基质间相互作用、抗原加工与提呈、PI3K-Akt信号通路、PPAR信号通路、补体与凝血级联反应等。VEGF-VEGFR2信号通路激活是噪声暴露后耳蜗血管纹微血管通透性改变的重要机制,噪声暴露后VEGF 通过与 VEGFR2 结合后激活酪氨酸激酶 Src 从而调控内皮细胞间黏附连接蛋白 VE-cadherin 的表达，引起血-迷路屏障通透性增加。噪声可以激活NF-κB炎症反应通路，活化基底膜巨噬细胞表达CD38蛋白，通过激活炎症复合体NLPR3，介导了内耳炎症和毛细胞的凋亡，通过药物抑制其过程中的关键分子NLRP3和CD38减轻内耳炎症反应可以保护听力。衰老过程可引起血管纹血-迷路屏障损伤并发生退行性变，免疫荧光染色观察对比血管纹形态学变化，发现老年小鼠血管纹毛细血管数量减少、密度降低，周细胞（PCs）数量显著减少、覆盖密度明显降低，PCs与毛细血管物理接触减少。本研究探索比较了耳蜗，尤其是血管纹在正常和病理情况时的蛋白和形态学差异，从而阐明血迷路屏障的功能及其调控机制。下一步，我们将以研究中发现关键蛋白质分子为切入点，深入推进靶向药物抑制内耳炎症的研究，进而从临床上进行老年性聋和噪声性聋病的药物防治。