自噬调节肺泡II型上皮细胞EMT过程对特发性肺纤维化的作用与机制研究

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive production of interstitial fibrous connective tissue caused by abnormal tissue injury and repair. Autophagy has been turned out to be a key role in tissue injury and repair homeostasis through its regulation of cell repair, survival or apoptosis in various diseases. Our preliminary study demonstrated that autophagy might be inhibited in IPF patients' lung tissue while the degree of pulmonary fibrosis induced by bleomycin (BLM) in mice was alleviated with enhanced autophagy by rapamycin. The mechanisms of these phenomenons remain unclear. The project intends to establish TGF-β1 induced EMT model in alveolar type II epithelial cell line treated with autophagy activator, inhibitor and siRNA transfection, observe the changes of cell morphology and markers, detect signaling molecules of Smad pathway, pro-fibrotic factors and extracellular matrix secretion and also the effect on cell apoptosis, in order to explore how autophagy regulates EMT in alveolar type II epithelial cell and underlying mechanisms. Pulmonary fibrosis model in vivo induced by BLM will be established to analyze the relevance between autophagy and EMT of alveolar type II epithelial cell in different stages; also through exogenous administration of inhibitor, activator and siRNA transfection in trachea, effect of autophagy regulation in EMT and pulmonary fibrosis will be further clarified. This study will further reveal the mechanisms of abnormal tissue injury and repair in IPF and provide theoretical and experimental support for clinical treatment and drug research in future.

特发性肺纤维化（IPF）的主要特征是损伤与修复异常导致的间质纤维组织过度增生。自噬通过调节细胞修复或死亡在多种疾病的损伤与修复中发挥重要作用。我们前期研究发现，IPF患者肺间质细胞中自噬可能存在抑制，通过雷帕霉素增强自噬可减轻肺纤维化，但机制尚不明确。本项目拟体外建立TGF-β1诱导肺泡II型上皮细胞EMT模型，观察细胞形态与表面标记变化，Smad通路相关信号分子表达，促纤维化因子与细胞外基质分泌及对细胞凋亡的影响，探索自噬对EMT的调控作用与机制。建立博莱霉素（BLM）诱导的肺间质纤维化动物模型，观察不同阶段肺泡II型上皮细胞自噬与EMT变化的关联；并针对不同阶段，气道内给予自噬增强、抑制剂及siRNA体内转染调节自噬水平，研究自噬调节EMT在肺间质纤维化中的作用。本研究将进一步揭示IPF损伤与修复异常的内在机制，为临床防治与新药开发提供理论和实验基础。

特发性肺间质纤维化（IPF）是一种慢性、不断进展的肺泡损伤和肺纤维化为特点的慢性、致死性疾病。IPF目前病因不明，吸烟以及环境因素如粉尘、硅和感染等在疾病的发生发展中均可能具有一定作用，而上皮细胞的损伤与修复异常是其重要的病理生理基础。既往研究证实了自噬、上皮细胞-间充质细胞转化（EMT）等参与了IPF的发生发展。IPF患者肺组织存在铁代谢异常，吡非尼酮可通过与铁形成复合物从而发挥治疗肺纤维化的作用。这些均暗示肺部铁稳态和铁超载在IPF发病机制中具有潜在作用，但其中具体机制尚无研究明确。本项目利用博莱霉素建立小鼠肺间质纤维化模型，明确了肺泡II型上皮细胞在早期炎症反应过程中存在自噬活性增加；体外利用MLE-12细胞系建立小鼠肺泡II型上皮细胞铁代谢异常模型，证实了细胞内铁浓度增加可激活细胞内氧化应激反应，增强肺泡II型上皮细胞自噬活性，诱导其向间充质细胞转化，参与纤维化形成；调控细胞自噬可有效抑制肺泡II型上皮细胞的EMT过程，并减少细胞外基质的分泌。这一结果为IPF的发病机制提供了新观点，提示在IPF发病过程中不同细胞具有不同的自噬活性变化，铁超载诱发的细胞氧化应激反应增加，可加重组织炎症反应，诱导自噬并促进EMT，且自噬在其中扮演了调控性角色。这也为IPF炎症早期的治疗提供了新思路。