分泌型金属蛋白酶CLCA在哮喘气道重塑中的作用及机制

Asthma is a common multiple chronic respiratory disease. Airway remodeling is one of the main pathological features of asthma, including epithelial remodeling, airway smooth muscle remodeling and extracellular matrix remodeling. As far as we known CLCA protein family is a molecular biomarker for asthma, its main function is promoting mucus hypersecretion. Recent studies report CLCA functions as self-cleaving secreted metalloprotease, and controls calcium activated chloride channel (CaCC) activation. Our previous studies showed that mClCA4 is not only expressed abundantly in airway epithelium and basement membrane, but also less in airway smooth muscle in asthmatic mice, suggesting that CLCA may have a close relationship with airway remodeling. This project aimed to disclose the effects of CLCA on asthmatic mice by using mCLCA recombinant proteins and mCLCA siRNA lentiviruses through studying on the following aspects: 1) whether dose CLCA induce proliferation and epithelial-mesenchymal transition in airway epithelial cells; 2) whether dose CLCA induce proliferation of airway smooth muscle cells; 3) whether dose CLCA induce ECM deposition through an imbalance of the MMP-9/TIMP-1 molar ration. This research will reveal the functions and mechanisms of CLCA mediating airway remodeling in asthma, and provide a theory evidence targeting CLCA in asthma therapy.

哮喘是一种常见的多发性慢性呼吸道疾病。气道重塑是哮喘的重要病理特征之一，包括上皮、平滑肌和胞外基质重塑等。已知CLCA蛋白是哮喘疾病的分子靶标，在哮喘中主要作用是促进黏液高分泌。近期的研究发现，CLCA蛋白是一种分泌型金属蛋白酶，具有自我剪切能力，可调控钙激活氯离子通道（CaCC）活性。我们的预实验结果显示mCLCA4不仅在哮喘气道上皮细胞和基底膜中大量表达分布，而且在平滑肌细胞中亦有少量表达分布，这表明CLCA与哮喘气道重塑可能密切相关。本研究通过制备mCLCA重组蛋白和siRNA-mCLCA慢病毒分别从在体和离体水平展开以下研究内容：1)CLCA能否诱导气道上皮细胞增殖和上皮-间质转化；2)能否诱导气道平滑肌细胞增殖；3)能否通过调节MMP-9/TIMP-1比例从而介导ECM的代谢失衡。本研究将揭示CLCA在哮喘气道重塑中的作用机制，为以CLCA为靶标干预治疗哮喘疾病提供理论依据。

CLCA蛋白家族与哮喘疾病存在密切的联系，已知其与粘液高分泌有关。本项目试图探索CLCA是否还参与哮喘气道重塑。利用mCLCA特异性抗体干预和构建的mCLCA-EGFP重组载体分别在动物活体水平和离体细胞水平检验mCLCA对气道上皮细胞增殖、上皮-间质转化以及细胞外基质代谢的作用。研究结果表明：1. mCLCA1-5基因在哮喘小鼠主气道、肺外支气管、肺内支气管以及肺叶组织中都不同程度上调，其中以mCLCA3基因上调倍数最为显著。2.哮喘小鼠肺叶组织的转化生长因子β1 (Tgfb1)和受体1 (Tgfbr1)基因表达显著高于正常小鼠。在细胞水平转染mCLCA-EGFP重组载体的支气管上皮细胞（16HBE）中亦发现Tgfb1和Tgfbr1基因表达上调。3.利用测序技术检测分析哮喘小鼠和正常小鼠肺叶组织的表达谱，通过RT-PCR技术验证发现Timp1、Mmp8、Mmp12、Mmp13、Mmp14、Mmp19、Adam8、Adamts4这8个细胞外基质代谢相关基因在哮喘小鼠肺叶组织中显著上调，Timp1和Mmp13的表达倍数较高。另外Timp1在哮喘小鼠肺叶组织的蛋白表达水平明显高于正常小鼠，而Mmp9蛋白水平未表现出显著差异。因此哮喘小鼠肺叶组织的Mmp9/Timp1比值明显低于正常小鼠。通过mCLCA抗体干预哮喘模型，我们发现mCLCA3和mCLCA4抗体可以显著降低Timp1的表达，而对Mmp9的表达没有影响。在细胞水平转染mCLCA-EGFP重组载体可诱导Timp1的表达。4.在细胞水平转染mCLCA-EGFP重组载体可诱导表皮生长因子受体(Egfr)的表达，并促进细胞增殖。因此，通过本项目研究证实mCLCA可促进气道上皮细胞发生上皮-间质转化、调节细胞外基质代谢、促进上皮细胞增殖。从而为以CLCA为靶标干预治疗哮喘疾病提供理论依据。