钙蛋白酶在肺间质纤维化上皮-间质转化过程中的作用及其信号传导机制研究

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown pathogenesis. Epithelial-mesenchymal transformation (EMT), an essential source of fibroblast, has been proved to be induced by TGFβ1 through activating EGF-ERK/MAPK siganl pathway. In our previous study, we found that the expression of calpain in lungs of patients with IPF was higher than that of normal people and TGFβ1 could induce EMT in A549 cells through up-regulating the expression of calpain. It was found that calpain can be activated by EGF-ERK/MAPK siganl pathway. CTGF, a downstream signal molecular of calpain was proved to be involved in the development of pulmonary fibrosis. Thus, we hypothesized that EMT can be induced by TGFβ1 through EGF-ERK/MAPK-Calpain-CTGF siganl pathway. We plan to investigate the expression of calpain and other molecular in lung tissue specimens. We also try to explore the detailed mechanisms of calpain in pulmonary fibrosis by transgenic mice model. In addition, we will explore the role of calpain in EMT and its signal pathway by type 2 alveolar epithelial cell culture in vitro. This research will improve the mechanism of IPF from a new perspective and provide a new target for the treatment of IPF.

特发性肺纤维化发病机制尚不清楚，上皮-间质转化（EMT）是肺成纤维细胞重要来源。研究表明TGFβ1 可通过激活EGF-ERK/MAPK途径诱导EMT发生。我们前期研究发现：特发性肺纤维化患者肺组织中钙蛋白酶表达显著上调；TGFβ1通过上调钙蛋白酶诱导了肺泡上皮细胞来源的A549细胞发生EMT。研究发现钙蛋白酶可被EGF-ERK/MAPK途径激活，钙蛋白酶的下游信号分子CTGF也参与了肺纤维化过程。据此，我们假设EGF-ERK/MAPK-钙蛋白酶-CTGF 是TGFβ1诱导EMT的信号通路之一。本课题拟通过收集临床肺组织标本，研究钙蛋白酶及信号通路分子的表达；通过转基因小鼠及建立肺纤维化动物模型，研究钙蛋白酶在肺纤维化过程中的作用及机制；通过肺泡Ⅱ型上皮细胞培养及干预研究进一步探讨钙蛋白酶在EMT中的作用及其信号传导机制。从而丰富特发性肺纤维化发病机制，并为特发性肺纤维化提供新的治疗靶点。

特发性肺纤维化（IPF）发病机制尚不清楚，上皮-间质转化（EMT）是肺成纤维细胞的重要来源之一。有文献表明TGFβ1可通过激活EGF-ERK/MAPK途径诱导EMT发生。我们前期研究发现IPF患者肺组织中钙蛋白酶（calpain）表达显著上调；TGFβ1可通过上调calpain诱导肺泡上皮细胞来源的A549细胞发生EMT。另有文献研究发现calpain可被EGF-ERK/MAPK途径激活，calpain的下游信号分子CTGF也参与了肺纤维化过程。据此，我们假设EGF-ERK/MAPK-calpain-CTGF是TGFβ1诱导EMT的信号通路之一。我们进一步研究发现：.1.与人正常肺组织相比，IPF患者肺组织中calpain和CTGF的表达明显上调，上皮细胞标志物E-cadherin的表达显著下调；.2.博来霉素（BLM）可以成功诱导小鼠发生肺纤维化，TGFβ1可以诱导A549细胞发生EMT；MEK抑制剂U0126和Calpain抑制剂PD150606均可减轻小鼠肺纤维化的程度；MEK抑制剂U0126可以显著降低小鼠肺组织及A549细胞中calpain的表达；同时MEK抑制剂U0126和calpain抑制剂PD150606均可抑制小鼠肺组织及A549细胞中的EMT过程。这些结果表明抑制ERK-calpain通路可能通过抑制EMT从而减轻肺纤维化；.3.MEK抑制剂U0126和calpain抑制剂PD150606均可抑制小鼠CTGF的表达。综合上述部分结果，研究表明ERK信号通过calpain调控CTGF参与肺纤维化的形成；.4.TGFβ1抑制剂SD208可通过抑制calpain的表达来减轻小鼠肺纤维化。综合上述部分结果，研究表明抑制TGFβ1-calpain通路可能通过抑制EMT从而减轻肺纤维化。.综上，我们的研究结果表明：抑制ERK-calpain及TGFβ1-calpain通路可能通过抑制EMT减轻小鼠肺纤维化程度，因此calpain可能是IPF未来的治疗靶点。