基于新型抗IPF分子Shp2基因敲除的自发肺纤维化小鼠模型建立与分析

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology with few current therapeutic options. Ideal animal models for pulmonary fibrosis are very limited, and Bleomycin (BLM) is a commonly used chemical agent to induce fibrosis in mouse, but it remains controversial due to the lack of several pathological features of IPF. Recent studies demonstrated that lung epithelial injury is critical to the initiation of pulmonary fibrosis. Phosphatase Shp2 is believed to be an intrinsic protective factor in alveolar lungs. Applicant’s studies previously showed that loss of Shp2 results in alveolar injuries and persistent alternative-activated macrophages in lungs. Mice with Shp2 deficiency develop spontaneous fibrotic alterations. These phenotypic observations received increasing attention from international experts in the field of pulmonary fibrosis. Most recently, clinical studies confirmed these mouse-based evidences, and found that a marked reduction of Shp2 protein in lungs collected from IPF patients, suggesting Shp2 is a novel anti-fibrotic regulator in pulmonary fibrosis. Based on these, this proposal aims to construct newly-developed mouse model for pulmonary disease, including 1) optimization of new strategies targeting Shp2 gene deletion (Shp2fl/-) for improving technique efficacy and stability, 2) comparative studies on modeling features, compatibility and IPF pathological indicators between new Shp2 deletion model and currently-used BLM model;3) further investigation on potentials of new disease model in the field of IPF etiology and anti-IPF drug studies. This proposal is expected to provide theoretical basis and experimental evidences, which is beneficial to the development of a novel animal model for pulmonary fibrosis.

特发性肺纤维化(IPF)病因不明，无有效治疗手段，也缺乏合适疾病动物模型。常用的博莱霉素(BLM)诱变小鼠模型难以反映IPF特征。肺上皮损伤是诱发纤维化的关键因素，磷酸酶Shp2是肺泡主要的内源性保护因素，申请人前期研究发现Shp2缺失导致肺泡组织损伤，肺泡巨噬细胞持续II型活化，肺上皮Shp2敲除小鼠表现自发肺纤维化特征，该表型获国外同行高度重视，最新临床验证并发现IPF患者肺组织Shp2显著减少，认为Shp2是一个全新抗IPF蛋白。基于此，并结合最新研究进展，项目聚焦模型构建与分析，研究包括：1）优化基因敲除方案，基于Shp2fl/-遗传背景，构建新的诱导式条件性敲除系统，提高敲除效率和稳定性；2）对比Shp2不同敲除方式小鼠，并与BLM模型就建模参数、兼容性和IPF相似性进行比对分析；3）探讨新模型在IPF机制及药物评价的适用性，预期有望为全新IPF模型研发提供理论基础与实验依据。

上皮损伤及炎症微环境调控在肺纤维化重构进程发挥关键作用。Shp2作为肺泡上皮损伤诱导纤维化的重要内源因素之一，上皮敲除Shp2可形成自发的小鼠肺纤维化表型，同时研究发现Shp2在肺泡上皮稳态调控以及巨噬细胞II型活化调控发挥关键作用，是肺纤维化重要的保护因子。我们研究已揭示肺泡微环境Shp2信号复合体调控肺纤维化的相关分子机制，阐明靶向敲除新型抗纤维化分子Shp2，构建肺纤维化疾病模型具备可行性和重要临床价值。我们利用本实验室构建的自发性及化学诱导肺纤维化模型、条件性敲除Shp2工具小鼠以及新建立的辐射诱导肺纤维化模型，明确了Shp2在肺泡上皮细胞、巨噬细胞、血管内皮细胞以及成纤维细胞等多种效应细胞中对纤维化的差异调控，不但为肺纤维化研究提供了新的实验模型和评价手段，也解析了磷酸酶Shp2 调控肺部微环境的复杂作用机制。首先，我们基于临床研究需求成功建立了辐射诱导肺纤维化小鼠模型，并以此为基础探讨了肺血管内皮细胞这一新的肺纤维化效应细胞中Shp2的新病因角色和靶向价值。同时基于肺部复杂的病理微环境，我们的研究发现了以Notch旁分泌信号为基础的内皮-巨噬相互作用，和以胞外囊泡为基础的上皮-巨噬相互作用对于肺部炎性损伤与纤维化重构的病理调控。在致病机制方面，一方面我们发现Shp2可以在肌成纤维细胞中通过新的纤维化调节Gremlin2-TGFβR1途径实现对肺纤维化的正反馈调控，同时我们还解析出拟素化翻译后修饰依赖于对Shp2构象及活化的调控参与肺纤维化病理进程，这些基于基因修饰小鼠及其疾病模型的关键研究数据为我们深入阐释IPF关键致病因子Shp2蛋白功能精准解析提供了理论依据，同时有望为肺纤维化疾病模型及其病理机制探索提供重要实验数据。