TGF-β/Smads信号通路调控线粒体自噬参与饲鸽者肺发生肺纤维化的分子机制研究

Pigeon breeder's lung(PBL) belongs to interstitial lung disease which is seriously harmful to the health of the Pigeon breeders. The study both domestic and aboard has found that TGF-β/Smads signaling pathway is involved in oxidative stress injury, which is one of the mechanisms of pulmonary fibrosis. Reactive oxygen species (ROS) can damage the mitochondrial membrane, and it can be removed by autophagy itself. The research group found that Smad3 gene hypomethylation and high expression of TGF-β and Smad3, which suggested that TGF-β/Smads signaling pathway is involved in pulmonary fibrosis in PBL. Therefore, we assumed that TGF-β/Smads signaling pathway by regulating mitophagy leads to the generation or clear anomaly of ROS and further promptes pulmonary fibrosis. This research will explore the expression of mitophagy markers in patients with PBL and detect characteristics of illed cells by pathological method. We will use Smad3 gene knockout mouse to construct the mouse animal model to study the effect of TGF-β/Smads signaling pathway on mitophagy, oxidative stress and pulmonary fibrosis, and we will construct a cell model involved in pathogenesis to further elucidate the molecular mechanism of TGF-β / Smads signaling pathway in lung fibroblast and alveolar epithelial cells regulating mitophagy involved in the pathogenesis of pulmonary fibrosis in PBL in order to provide a theoretical basis for the prevention and treatment of such disease.

饲鸽者肺属于间质性肺疾病，严重危害饲鸽者人群的健康。国内外研究发现TGF-β/Smads信号通路参与氧化应激损伤，是发生肺纤维化的机制之一；活性氧簇（ROS）可损伤线粒体，并通过自噬途径进行清除。我们发现Smad3基因低甲基化及TGF-β、Smad3高表达，提示TGF-β/Smads信号通路参与饲鸽者肺患者发生肺纤维化进程。因此，提出假说：TGF-β/Smads信号通路可能通过调控线粒体自噬导致ROS产生或清除异常，促使肺纤维化发生。本课题拟研究饲鸽者肺患者线粒体自噬标记物表达，用病理学方法检测病变细胞特点；用Smad3基因敲除大鼠构建动物模型，研究TGF-β/Smads信号通路对线粒体自噬、氧化应激及肺纤维化的影响；构建参与发病的细胞模型，进一步阐明肺成纤维细胞、肺泡上皮细胞内TGF-β/Smads信号通路调控线粒体自噬参与饲鸽者肺发生肺纤维化的分子机制，为防治此类疾病提供理论依据。

本课题首先分析饲鸽者肺患者临床特点，并对饲鸽者肺功能的观察研究，探讨饲养鸽子会对肺功能产生影响。饲养鸽子时间越长，对肺功能的影响越大，为早期发现饲养鸽子人群肺功能改变提供依据，对其早期发现、早期预防具有重要意义。同时构建不同病理时期饲鸽者肺疾病动物模型，首次构建了纤维化期动物模型，以动物模型为基础，通过Smad3基因敲除组、干预自噬，利用大鼠动物模型研究纤维化过程中TGF-β/Smads信号通路与线粒体自噬的关系，包括：1、观察各组病理学差异。2、肺组织线粒体的提取并观察线粒体形态。3、应用蛋白免疫印迹技术检测线粒体自噬及TGF-β/Smads信号通路及相关通路关键蛋白的表达量。4、用荧光定量PCR 技术检测各组肺纤维化相关因子mRNA的表达。5、建立饲鸽者肺细胞模型并进行细胞培养。6、免疫细胞化学方法检测LC-3，Beclin-1，TNF-α，TGF-β蛋白。7、DCFH-DA探针检测16M诱导ROS产生水平。研究发现：纤维化型饲鸽者肺大鼠肺组织中TGF-β1、Smad3、线粒体自噬因子LC3和Beclin1蛋白表达明显升高，说明TGF-β/Smad3和线粒体自噬参与饲鸽者肺的纤维化，抑制或诱导线粒体自噬抑制或促进TGF-β1、Smad3表达。同时阻断TGF-β/Smad3信号通路肺纤维化的模型中线粒体自噬因子Beclin1和LC3表达降低。从细胞模型验证，І、Ⅱ型肺泡细胞主要参与线粒体自噬，TGF-β1/Smad3信号通路通过调控成纤维细胞自噬参与饲鸽者肺的纤维化，初步探讨了TGF-β/Smads信号通路调控自噬参与饲鸽者肺发生纤维化的机制。研究结果对患者的纤维化机制提供了分子机制，对认识疾病的发病及纤维化机制，并设计治疗和干预策略具有重要的意义。