TGF beta 1在噪声损伤后耳蜗微循环PC细胞纤维化中的作用及机制研究

There are plentiful Pericytes (PC) in the inner ear microcirculation, but the physiological functions and pathological mechanisms of it are not clear. Many studies indicated that the fibrosis of PC might be the pathomechanism of lots of common disease (such as myocardial infarction, cerebral stroke and so on). Our previous study showed that PC protruded from the capillary wall and detached from it in the mouse after noise exposure, then the migrated PC relocated near the microvascular wall. So the permeability of capillary boosted, damage of blood labyrinth barrier occurred which led to the disorder of cochlear microenvironment. PDGF-BB signaling pathway played key role in this process. However, the state of migrated PC and the effect on microcirculation has been unknown. Therefore, thin and open vessel windows for intra-vital fluorescence imaging of mouse cochlear blood flow, 3D co-culture of strial vascularis cells, and other in vitro, in vivo models will be used to observe the morphology of PC and microcirculation before and after noise exposure. Related factors and the relationships between them will also be detected. The proposed studies are designed to investigate whether PC-to-myofibroblast transition is included in the pathological process of noise induced hearing loss, identify the main signaling system in it, especially focus on TGF beat 1. In order to develop the remedial therapy, the antagonist will be used to inhibit the process of PC fibrosis. A specific goal of this study is to determine whether cochlear PC from the neonatal mouse can be utilized as stem cells in repair of loud sound damaged vessels and for restoration of microcirculation and auditory function. The results generated from this proposal will not only advance our understanding of loud sound induced vascular damage but also lay the foundation for prevention and remedial therapy of it.

内耳微循环中有大量周细胞(Pericyte, PC)，但我们对其生理及病理机制却知之甚少。PC细胞的纤维化是很多疾病的病理基础，如心梗、脑梗等。申请人的前期研究发现噪声损伤后的小鼠内耳微循环中PC细胞会发生突起、脱落并迁移至血管周围，从而导致血迷路屏障破坏，引发内耳微环境紊乱。此过程主要由PDGF-BB及其信号传导通路介导。但迁移后PC细胞的情况及其对微循环的影响都尚未阐明。为此，我们计划应用小鼠活体耳蜗开窗、3D细胞培养等各种在体、离体方法来观察噪声前、后微循环和PC细胞的变化，各相关因子情况及其与周围组织的关系。明确在噪声致聋中是否存在PC细胞纤维化及其对微循环的影响，确定主要信号传导途径，重点关注TGF beta 1，并尝试应用拮抗剂来抑制此过程。确定新鲜幼鼠PC细胞是否可作为干细胞来修复受损的微循环并恢复听力。这不仅会加深我们对噪声致聋机制的认识，还可能成为研发防治手段的基础。

内耳微循环中有大量周细胞(Pericyte, PC)，但我们对其生理及病理机制却知之甚少。PC细胞的纤维化是很多疾病的病理基础，如心梗、脑梗等。本研究应用小鼠活体耳蜗开窗、3D细胞培养等各种在体、离体方法来观察噪声前、后微循环和PC细胞的变化。结果发现噪声损伤后的小鼠内耳微循环中PC细胞会发生突起、脱落并迁移至血管周围，转化成肌纤维母细胞，并使组织发生纤维化，造成微循环闭锁，从而导致血迷路屏障破坏，引发内耳微环境紊乱，最终导致听力损失。此过程主要由TGF-β1信号传导通路介导，噪声后2周的小鼠内耳血管纹组织中TGF-β1的mRNA和蛋白表达量显著增加。应用TGF-β1的特异性受体拮抗剂可减轻此转变的比例。离体培养的PC细胞实验也显示，在细胞培养基中逐渐增加TGF-β1活性蛋白的浓度，可以使PC-肌纤维母细胞的转变比例增加。3D细胞培养模型和小鼠活体实验表明，转变后的肌纤维母细胞可使大量的细胞外基质蛋白沉积于血管周围，包括胶原蛋白IV（Collagen IV）、Laminin等，逐渐导致组织发生纤维化改变，血管网络密度减低，功能退化，部分血管闭锁。我们还将新鲜幼鼠PC细胞和AAV1-VEGF-A转染后的PC细胞通过半规管注射的方法导入内耳，将其作为干细胞来修复噪声损伤的血管纹结构。通过小鼠活体耳蜗外侧壁开窗技术，我们观察到在噪声损伤后血管纹会出现血流量下降，血管密度降低。而干细胞和基因治疗后血流量会明显改善，同时蜗内电位和听力功能都有部分恢复。本研究的结果不仅进一步阐明了噪声导致的内耳微循环障碍及修复的具体机制，而且有助于我们开发能够应用于噪声性聋临床治疗的有效方法。这对其他内耳疾病（如老年性聋、药物性聋、突发性聋等）的微循环障碍发生机制和临床治疗研究也有一定的促进作用。