Calpain调控胸膜间皮细胞的增殖与迁移在胸膜及胸膜下纤维化发生中的作用

Pleural and subpleural disorders are very common in interstitial lung diseases (ILD). But the role of pleural mesothelial cells (PMCs) in the ILD was poorly understood. Mubarak et al reported parenchymal trafficking of PMCs in idiopathic pulmonary fibrosis which was published in European Respiratory Journal in 2012. The study suggested that PMCs should be very important in ILD, but more investigations are needed to reveal the detail mechanisms. In recent years, researchers revealed that calpain plays a pivotal role in regulating cell migration. Many factors inducing extracellular matrix remodeling via calpain was also confirmed recently. We previously found calpain mediated pulmonary arterial smooth muscle cells remodeling and pulmonary hypertension. In our preliminary experiments, we found PMCs migration into lung tissue via calpain in mice model of pulmonary fibrosis. In vitro, bleomycin inducing PMCs proliferation and migration through calpain was also confirmed. Taken together, we hypothesized that fibrotic factors induced increase in calpain activity, and then the activation of calpain induced PMCs proliferation and migration, as well as extracellular matrix remodeling, which is the key mechanism in pleural and subpleural fibrosis. In the project, we will do experiments using cell models and animal models, especially using calpain-4 knockout mice to confirm our hypothesis. The results of this project will be helpful for both understanding the mechanism and treating the disorders of fibrosis in ILD.

许多间质性肺疾病有明显的胸膜及胸膜下病变，其发生机制是否与胸膜间皮细胞直接相关呢？近年有报道发现特发性肺纤维化病人的胸膜间皮细胞向肺内迁移，提示胸膜间皮细胞可能在间质性肺疾病的发生中起到非常关键的作用。同时，近年的研究进展证实"钙蛋白酶(calpain)诱导细胞迁移"、"calpain促进细胞外基质重建"。申请者的研究已经证实calpain参与肺血管重建，而且预实验提示"鼠肺纤维化中calpain介导胸膜间皮细胞肺内迁移且与纤维化病灶相关；体外培养中calpain介导胸膜间皮细胞增殖与迁移"。鉴于此，申请者拟进一步从分子、细胞及整体水平（包括利用calpain基因敲除小鼠）展开研究，以证实"胸膜间皮细胞在致病因子的作用下，由calpain介导，发生细胞增殖并向肺内迁移，参与细胞外基质重建，诱导胸膜及胸膜下纤维化发生"的创新性假说，为间质性肺疾病的防治探索新策略。

许多间质性肺疾病有明显的胸膜及胸膜下病变，其发生是否与胸膜间皮细胞直接相关值得探讨。已有报道发现特发性肺纤维化病人的胸膜间皮细胞向肺内迁移，提示胸膜间皮细胞可能在间质性肺疾病的发生中起关键作用。同时，研究进展证实“钙蛋白酶(calpain)诱导细胞迁移”、“calpain促进细胞外基质重建”。项目申请时，我们团队的研究已经证实calpain参与肺血管重建，而且预实验提示“鼠肺纤维化中calpain介导胸膜间皮细胞肺内迁移且与纤维化病灶相关”。因此，为了证实胸膜间皮细胞在胸膜及胸膜下纤维化发生中的作用与机制，我们开展的主要研究内容包括：①选取了博莱霉素、TGF-β1及PDGF作为致纤维化因子，观察他们活化calpain、介导胸膜间皮细胞增殖、迁移、分泌胶原的变化；②研究了calpain促进细胞增殖及迁移的具体机制，探索是否调控粘着斑相关蛋白、细胞连接蛋白、细胞骨架蛋白而促进细胞移行；③整体水平上利用本研究组拥有的calpain基因条件敲除小鼠，在体观察胸膜间皮细胞迁移、胸膜及胸膜下纤维化的发生情况。.项目取得的重要结果包括：促纤维化因子TGF-β1、博莱霉素诱导胸膜间皮细胞内calpain活化、并导致胶原合成增加与细胞增殖与定向迁移；TGF-β1、博莱霉素诱导胸膜间皮细胞骨架蛋白改变而促进细胞迁移，calpain介导粘着斑激酶(focal adhesion kinase, FAK)活化是其可能的机制；calpain活化与TGF-β1高表达之间相互促进，诱导肺纤维化发生；动物整体水平上，calpain抑制剂、calpain基因敲除减少胸膜间皮细胞肺内迁移并减轻小鼠肺纤维化的发生。项目对肺纤维化尤其胸膜下肺纤维化的发生提出了一个新的解释，提出了calpain介导肺纤维化的新理论；同时为临床上防治肺纤维化提供了一个新的靶点，有望使用药物调控calpain活性从而预防或治疗肺纤维化。