ATF3在肺成纤维细胞间质细胞转化中的作用及调控机制

Pulmonary fibrosis is a progressive disease which is characterised by widespread fibrotic damage of the interstitial tissue of lung.It can cause severe complication such as respiratory failure and s chronic pulmonary heart disease。Currently， no effective drugs available that can control or slow the fibrotic process，and the patients median survival rate is merely 3 to 5 years after diagnosis.Though the pathophysiology of the disease Idiopathic pulmonary fibrosis is a progressive disease which is characterised by widespread fibrotic damage of the interstitial tissue of lung.It can cause severe complication such as respiratory failure and s chronic pulmonary heart disease。Currently， no effective drugs available that can control or slow the fibrotic process，and the patients median survival rate is merely 3 to 5 years after diagnosis.Though the pathophysiology of the disease still remains not fully understood， it is general accepted that the myofibroblast is the pivotal player，which contributes to the excess deposition and remodeling of the extracellular matrix。the generation of myofibroblast has many origins，recently，it is revealed that vascular endothelial cell can undergo the process of endothelial–mesenchymal transition in some specific conditions such as TGF-beta is presented，and acquires the matrix-producing myofibroblastic features，and it is unfold that EMT is an important source of myofibroblast in fibrotic disease. yet the particular mechanism that control this process is still vague. ATF3nvolves cellular processes such as actin dynamics, permeability、motility, , cell-cell adhesion、proliferation and apoptosis. lately some proofs showed that PAK1 is also participate in transformation of varous source of cells，yet whether it is involved in the EMT remained unsettled。 Thus we here raise the hypothesis：ATF3 signaling is a novel player in the EMT，and hydroxychloroquine can down-regulate the activity of the ATF3，diminish the gerneration of myofibroblast，thereby exert the Pharmacological effect of treating pulmonary fibrosis.to verify the hypothesis，We aim to interfere the human umbilical vein endothelial cells by TGF-beta and hydroxychloroquine，and explore the change of the adhesion molecules、rearrangement of cytoskeleton，expression of alpha-sma、fibronectin、MMPs、integrin and collagen. BY establishing the Bleomycin induced pulmonary fibrosis in mice,we can observe the role that ATF3 participate in EMT inexperimental pulmonary fibrosis. Though which, we are to provide a thorough new understanding for the pathological mechanism，and development of novel medical treatment in this disease.

结缔组织病相关肺纤维化病情发展的关键是人肺成纤维细胞（HLF）在TGF-β1刺激下间质转化为肌成纤维细胞，其迁移侵袭能力增强并分泌大量胶原导致肺结构破坏，但其机制尚不清楚。近年发现ATF3（Activating Transcription Factor 3）是调控细胞迁移、侵袭的重要因素；参与调控ATF3的相关蛋白可能通过激活Gli1蛋白促进细胞迁移。我们前期发现ATF3及Gli1蛋白在TGF-β介导的HLF间质转化中表达增高，且均在肺纤维化患者中升高，敲除ATF3则可抑制HLF骨架重构。因此基于以往的工作基础，本课题拟研究ATF3对HLF间质转化、迁移和侵袭的调节作用及其是否通过Gli1蛋白介导；并利用博来霉素诱导肺纤维化和ATF3条件基因敲除小鼠模型；观察体内调控ATF3相关蛋白表达对HLF间质转化的影响及机制，同时了解干预后对肺纤维化病情的改善作用，旨在为肺纤维化提供新型的治疗靶点。

肺纤维化是结缔组织病常见的疑难并发症，发掘其病理机制和靶点是当前治疗的当务之急。研究表明ATF3在皮肤纤维化里起促进作用。因此申请者提出了ATF3通过促进成纤维细胞间质转化从而参与肺纤维化进程的假说。在基金资助阶段，申请者完成了基金申请书中的主要研究计划，阐明了ATF3通过上调经典转化Smad3通路从而上调人肺成纤维细胞的各项细胞功能和间质化标志，并重点讨论了抑制该通路后对纤维化的减轻。目前研究结果包括：1、ATF在体外博来霉素诱导的肺纤维化小鼠中升高，与成纤维细胞标志共定位；2、ATF3经Smad3通路诱导成纤维细胞表达间质标志alpha和骨架蛋白F-actin重构，通过敲减ATF3可有效抑制TGF诱导的细胞转化；3、敲减ATF3可有效抑制TGF诱导细胞迁移、侵袭、增殖、分泌等能力；4、吡非尼酮等药物可通过抑制ATF3升高从而抑制细胞转化、减轻肺纤维化。科学意义：本课题明确了ATF3通过诱导成纤维间质转化从而参与肺纤维化的过程，为了解肺纤维化的机理和靶点干预提供新的线索和认识。