肺微环境粘附分子谱对气道上皮／间质修复平衡点调控

The proposal hypothesize that imbalance between epithelial repair and mesenchymal repair possibly contribute the innate cause of pulmonary fibrosis or airway remodelling, and the control mechanism of this repairring equation may involve the interaction between epithelial cells and mesenchymal cells which might be regulated by expression spectrum of adhesive molecules in the pulmonary microenvironment. The project plan: ① A stress injury animal model will be induced on a mouse with integrinβ4 conditional knockout in airway epithelium that has been created in our laborytory, and on this model, ventilation function,lung architecture,some phenotype marker of epithelial-mesenchymal transition (EMT) or mesenchymal-epithelial transition (MET),and their correspond relation with three major kind of adhesive molecules will be observed. ② On cellular level, both airway epithelial celline and pulmonary fibroblast celline with different expression level of three major adhesive molecules will be constructed, and proliferation,repairring index, EMT or MET marker, and difference in collagen synthesis spectrum also be evaluated.③ In a coculture system composed by airway epithelial cells and pulmonary fibroblasts, interaction between two cellline with different adhesive molecules expression spectrum is planed to be investigated and those relevant secretory medium will be idientified.④ From data analysis,an optimal compose of adhesive molecules which can most effetively control the balance between epithelial and mesenchymal repair will be found, and some transinfect system then be designed according to this optimal compose to do functional confirmation. This study will bring a new sight to lung injury repair,pulmonary fibrosis and airway remodelling, and try to seek for new target from adhesive molecules in pulmonary microenvironment for therapy of above lung diseases.

申请者提出上皮修复和间质修复的失衡可能是肺纤维化和气道重构的内在原因，粘附分子表达谱及其调控的上皮细胞与间质细胞的相互作用构成平衡调控机制。本项目拟：①利用自主构建的气道上皮整合素β4条件性剔除小鼠，建立损伤模型，观察肺功能、结构、上皮细胞与间质细胞的EMT和MET表型标记，及其与三种主要粘附分子表达谱的对应关系；②在细胞水平，构建3种主要粘附分子不同表达水平的气道上皮细胞系和肺成纤维细胞系，检测各组细胞的增殖、修复指数、EMT和MET表型、胶原合成谱等差异；③利用气道上皮细胞与肺成纤维细胞共培养体系，观察在不同粘附分子表达谱下两种细胞的相互作用，确定相关的分泌型介质；④通过数据分析，寻找有效实现上皮修复与间质修复平衡控制的最佳粘附分子表达水平组合，构建最佳组合转染体系，进行功能验证。该研究将创新肺损伤修复、肺纤维化、气道重构的研究思路，寻求以粘附分子作为治疗上述疾病新靶点的可能性。

肺和气道是外部环境与机体内环境相互作用的第一道界面，经常遭受来自内外环境的应激刺激或损伤。本项目在动物模型整体水平和细胞水平两个部分阐明肺损伤微环境中粘附分子谱对上皮修复和间质修复平衡点的调控，对慢性损伤后肺纤维化和气道重构进行新的机制描述。研究结果如下：①臭氧应激可以诱导小鼠支气管肺上皮细胞向间质细胞转化，在臭氧应激的小鼠EMT模型中整合素β4的变化与肺组织EMT和MET的表型相关；②在HDM或OVA诱导的哮喘小鼠模型，肺组织EMT表型与粘附分子整合素β4、CTNNAL1及ICAM-1的表达相关；③在臭氧应激的细胞EMT-MET模型上，观察到损伤程度与EMT-MET的转换直接相关，随着臭氧损伤刺激的增强，EMT标记分子表达、细胞骨架重组和力学改变的恢复程度持续降低。臭氧损伤后，粘附分子整合素β4、ZO-1, CTNNAL-1, ICAM-1的表达水平的变化与HBE-EMT分子表型显著表达及细胞骨架重构密切相关；④细胞水平实验证实粘附分子CTNNAL1过表达下调人支气管上皮细胞分泌TGF-β1，抑制臭氧应激后人支气管上皮细胞EMT发生，有助于受损细胞维持上皮表型；⑤粘附分子E-cadherin通过TGFβ1-Smad信号通路调控减弱人支气管上皮细胞臭氧应激诱导的EMT的发展，促进MET，即促进间质向上皮细胞的转归，促进修复完成。以上结果提示，损伤时上皮细胞发生EMT表型转变可能是上皮细胞应答损伤发生修复的一种反应形式，当完成细胞增殖和迁移时，细胞表型再次由间质细胞表型向上皮细胞回归（MET），最终恢复上皮的结构和功能。当损伤刺激较强时，EMT不能被及时限制或MET不能发生，则修复平衡不能维持，是发生纤维化或重构的基础。粘附分子参与肺和气道损伤修复中上皮／间质修复平衡控制，粘附分子表达失平衡可能是肺应激损伤后气道重塑和肺纤维化形成的启动机制。本研究拓展了对支气管肺上皮粘附分子表达平衡生物学意义的认识，为肺损伤修复及其转归机制解释和临床干预提供重要的线索。