【作者】 陈玲；

【导师】 韩晓冬； 陈惠君；

【作者基本信息】 南京大学 ， 基础医学， 2019， 硕士

【摘要】 特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)是一种慢性、进行性、不可逆转的一类肺间质疾病,病因不明,主要表现为纤维灶的出现,肺泡结构破坏,大量细胞外基质沉积。临床表现为进行性呼吸困难,肺功能下降,病情一般持续恶化,最终因呼吸衰竭而死亡。IPF是一种老年性疾病,发病率与年龄相关,随着社会老年化进程加快,近年来IPF发病率呈不断上升趋势,且目前并无有效的药物治疗手段,确诊后的中位生存期只有3-5年。IPF的发病机制目前尚不清楚,已经明确的是成纤维细胞的过度增殖是导致肺纤维化最直接的原因。研究表明Wnt/β-catenin信号通路在肺纤维化发生发展过程中发挥重要作用,激活该信号通路可诱导肺间质干细胞向成纤维细胞分化,促进肺泡上皮细胞向成纤维细胞转化,可导致成纤维细胞的增殖与活化。β-catenin在胞质内大量积累是Wnt/β-catenin信号通路激活的重要标志,在肺纤维化发生发展过程中有效控制β-catenin产生对于抑制其纤维化至关重要!在调控Wnt信号通路对IPF发生发展的长期研究中我们发现GSK-3β是否具备活性是β-catenin能否被泛素化降解的关键环节。组织型纤溶酶原激活剂(Tissue plasminogen activator,tPA)是丝氨酸蛋白酶家族中的一员,在以往对tPA的研究中,研究者主要关注的是tPA作为蛋白酶在凝血、纤溶稳态中所起的作用—tPA可以溶解纤维蛋白和调节细胞外基质。有研究表明,tPA除了作为蛋白酶在溶栓系统中发挥重要作用以外,还可以作为细胞因子参与到信号通路中,它能够作为一种细胞因子激活细胞增殖信号通路,导致GSK-3β磷酸化水平升高,活性受到抑制。因此,本研究中以tPA作为着眼点,探究tPA信号通路在特发性肺纤维化中的作用机制研究。经气道雾化给予博来霉素构建小鼠IPF模型,我们发现:1、在IPF小鼠肺组织中tPA的表达明显上升,且tPA信号通路处于激活状态,提示tPA可能在小鼠肺纤维化发生发展过程中发挥作用。2、tPA对小鼠原代成纤维细胞有增殖作用,tPA通过细胞膜上受体激活胞质内的Erk1/2蛋白,使下游p90RSK蛋白、GSK-3β蛋白磷酸化,促进Cyclin D1蛋白表达升高,促进成纤维细胞的增殖。另外,tPA通过抑制GSK-3β的活性,导致β-catenin表达升高,间接激活了 Wnt/β-catenin信号通路。3、在动物体内,tPA信号通路抑制剂PD98059能够降低GSK-3β磷酸化水平,一定程度地抑制Wnt/β-catenin信号通路,干预肺纤维化的发生发展。更多还原

【Abstract】 Idiopathic pulmonary fibrosis(IPF)is a chronic,progressive and irreversible fatal pulmonary disease of unknown etiology,which characterized by the appearance of fibroblast foci,deposition of a large number of extracellular matrix,accumulation of collagen and the destruction of alveolar structure.The severely affected patients exhibited an acute respiratory distress syndrome and decreased lung function,the condition continues on its course and the patients eventually die of respiratory failure.IPF is a geriatric disease,and its incidence is related to age.With the increasingly aging of the population,the incidence of IPF has been on the rise in recent years.Currently,there is no effective drug therapy,and the median survival after diagnosis is only 3-5 years.At present,the pathogenesis of IPF is unclear,but a lot of studies have shown that Wnt/β-catenin signaling pathway plays an important role in the development of pulmonary fibrosis,which can promote lung mesenchymal stem cells differentiation,induction of alveolar epithelial cells to fibroblasts and lead to fibroblast proliferation and activation.The large accumulation of β-catenin in the cytoplasm is an important marker of the activation of Wnt/β-catenin signaling pathway,and the effective control of β-catenin production during the occurrence and development of IPF is crucial.In the long-term study on the regulation of Wnt signaling pathway,we found that the activity of GSK-3β is a key to whether β-catenin can be ubiquitinated and degraded.Tissue plasminogen activator(tPA)is a member of the serine protease family.In previous studies,researchers mainly focused on the role of tPA as a protease in coagulation and fibrinolysis homeostasis,tPA can dissolve fibrinin and regulate extracellular matrix.Studies have shown that in addition to playing an important role in the thrombolytic system as a protease,tPA can also activate the cell proliferation signaling pathway as a cytokine,resulting in the inhibition of the activity of GSK-3βprotein in the signaling pathway.Therefore,this study we focus on tPA and explore the mechanism of tPA signaling pathway in idiopathic pulmonary fibrosis.We used bleomycin to establish IPF mouse model,first,we found that the expression of tPA in the lung tissue of IPF mice was increased,and the tPA signaling pathway was activated,suggesting that tPA may play an important role in the development of IPF.Second,tPA activates Erk1/2 protein in the cytoplasm through receptors on the cell membrane,phosphorylates downstream p90RSK protein and GSK-3β protein,increases the expression of CyclinDl protein,and promotes the proliferation of fibroblasts.In addition,tPA indirectly activates the Wnt/β-catenin signaling pathway by inhibiting the activity of GSK-3β,resulting in increased expression of β-catenin.Third,in vivo,tPA signaling pathway inhibitor PD98059 can reduce the level of GSK-3β phosphorylation,inhibit the Wnt/β-catenin signaling pathway to a certain extent,and intervene in the occurrence and development of IPF.更多还原