microRNA调控TGF-β1/Smad3信号通路对声带损伤纤维化的作用研究

Vocal fold injury often resulted in extracellular matrix disorderly deposition,and thus the fibrous scar formation, which caused irreversible dysphonia. How to slow or reverse the process of excessive fibrosis after vocal fold injury is the key to the treatment. In our previous study, we found that the fibroblasts are extremely hyperplastic, collagen-predominant extracellular matrix disorderly deposition and fibrous tissue hyperplasia after vocal fold injury. Our further study showed that the TGF-β1/Smad3 signaling pathway play an important role in the process of vocal fold fibroblasts over-activities and extracellular matrix abnormal secretion. However, how to regulate this signaling pathway in order to inhibit the process of excessive fibrosis need further study. For this purpose, we plan to set up the microRNA expression profile of vocal fold injury and to investigate the related microRNA of TGF-β1/Smad3 signaling pathway to study its regulation of vocal fold fibroblasts and extracellular matrix in vitro. Meanwhile, we design to construct microR-target smad3-GFP-overexpression lentivirus vector,and implant into the vocal fold injury site of animal model to explore its possible mechanism in the process of vocal fold fibrosis, to understand its significance for the repair and reconstruction of the vocal fold proper lamina, and provide a key intervention of the site for the visualization of the fibrotic process investigate the related microRNA of TGF-β1/Smad3 signaling pathway to study its regulation of vocal fold fibroblasts and extracellular matrix in vitro. Meanwhile, we design to construct microR-target smad3-GFP-overexpression lentivirus vector, and implant into the vocal fold injury site of animal model to explore its possible mechanism in the process of vocal fold fibrosis, to understand its significance for the repair and reconstruction of the vocal fold proper lamina, and provide a key intervention of the site for the visualization of the fibrotic process.

声带损伤引起固有层细胞外基质无序沉积，进而形成纤维化瘢痕，引起不可逆性发声障碍。如何减缓或逆转声带损伤后过度纤维化的进程是治疗的关键。我们前期研究发现，声带损伤后成纤维细胞增生极度活跃，以胶原为主的细胞外基质紊乱沉积；同时还发现TGF-β1/Smad信号通路参与调控声带修复及纤维化进程，Smad3可能是干预的关键靶点。因此，我们推测TGF-β1/Smad3信号通路可能在声带损伤后纤维化过程中起关键作用，通过microRNA调控该信号通路，有可能抑制或改变声带纤维化过程中成纤维细胞的过度激活及细胞外基质的异常分泌。为此我们拟建立损伤声带microRNA表达谱，筛选与该信号通路相关microRNA，研究其对声带成纤维细胞及细胞外基质的调控作用；同时利用基因转染过表达或敲减此种microRNA，探讨其在损伤声带纤维化进程中可能的作用机制，为探索声带损伤后纤维化的关键干预位点提供依据。

声带损伤引起纤维化瘢痕导致的发声障碍一直是研究的难点，如何减缓或逆转声带损伤后过度纤维化的进程是治疗的关键。本课题研究了损伤声带修复过程中不同时期的差异基因及相关信号通路变化，并探讨microRNA调控TGF-β1/Smad3信号通路在声带损伤后异常纤维化过程中可能的作用。首先进行了TGF-β对声带成纤维细胞活化作用的研究，运用基因芯片技术筛选出大鼠声带损伤后不同时期声带组织中的差异基因，利用生物信息学技术，探索声带损伤后不同时期修复过程中的主要生物学功能和信号通路,发现声带损伤后1周细胞活动最为活跃，与成纤维细胞增殖分化以及细胞外基质合成相关的细胞功能、信号通路上调明显，是纤维化形成的关键时期；声带损伤后8周仍存在免疫反应，可能持续促进声带纤维化的进程。在声带损伤修复过程中，发现催乳素在声带损伤修复过程中表达显著下调，且表达的变化与损伤修复过程高度相关，可能在声带损伤修复过程中起到关键作用。其次筛选出大鼠声带损伤修复过程中差异microRNA，探讨microRNA与差异基因的调控关系，寻找声带损伤修复过程中microRNA与基因可能的调控通路，发现miR-181-5p对催乳素mRNA的调控具有时间依赖性及剂量依赖性，并具有靶向调控催乳素 mRNA的特异性，提示miR-181-5p在声带损伤修复可能通过调节催乳素的表达影响声带损伤修复过程，催乳素和miR-181-5p可能成为声带损伤修复过程中的干预靶点。此外，本课题还对TGF-β1/Smad3通过下游SOCS3、JAK/STAT信号通路调控声带成纤维细胞活化的机制进行了探索，发现声带损伤后SOCS3表达减少，JAK2/STAT3信号通路被激活，SOCS3可能在声带损伤早期，通过下调JAK2/STAT3信号通路的转导来抑制声带成纤维细胞的过度激活。