整合素β4缺陷致肺发育不良及其机制

Our previous study found that integrin β4 was involved in the susceptibility to asthma. In our proposed project we plan to explore the role of integrin β4 in lung development and try to identify whether the defects of this molecule leads to Bronchopulmonary Dysplasia (BPD) by disturbing the interaction between bronchial epithelial cells and its surrounding extracellular matrix (ECM). We have already established an integrin β4 conditional knockout mouse model which can shut off the expression of integrin β4 in bronchial epithelial cells. We plan to use this animal model to observe the impacts of integrin β4 knockout on the developing lung tissue. We will investigate the alterations of lung tissue morphology, differentiation markers and major signaling pathways related to lung development. We will also observe the expressions of different components of extracellular matrix in lung tissue and explore the relationships between ECM formation of lung tissue and integrin β4. Meanwhile, we will construct airway epithelial cells with different expression levels of integrin β4 and observe the impacts of integrin β4 on the interactions between epithelium and ECM or between epithelium and mesenchymal cells.Our proposed study will provide evidence for the regulatory roles of integrin β4 on lung development by recognizing ECM environmental changes and interacting with ECM. It will also gain novel insights into mechanisms of lung development and identify new therapeutic targets of Bronchopulmonary Dysplasia (BPD).

本项目以申请者课题组前期发现的哮喘易感性相关粘附分子整合素β4为切入点，探讨整合素β4介导的上皮-基质相互作用在支气管肺发育不良中的作用。拟利用本课题组自主构建的气道上皮整合素β4可诱导性剔除小鼠模型，观察在胚胎期诱导整合素β4沉默对肺发育组织形态、肺分化标志性分子及肺发育相关主要信号通路的影响；观察整合素β4和肺基质主要成分的表达与时空分布及他们之间的相互关系。同时构建整合素β4不同表达水平的气道上皮细胞模型，以其基质配体层粘连蛋白5作为刺激因素，观察整合素β4对上皮-基质及上皮-间充质细胞相互作用的影响。论证气道上皮整合素β4通过识别、感受及影响基质环境对肺发育发挥调控作用。本研究将拓展粘附分子与肺发育内在联系的认识，寻求以整合素β4作为肺发育先天性疾病防治新靶点的可能，同时也有助于加深对成人获得性肺疾病的发生发展的理解。

本项目以申请者课题组前期发现的哮喘易感性相关粘附分子整合素β4为切入点，探讨整合素β4介导的上皮-基质相互作用在支气管肺发育不良中的作用。利用本课题组自主构建的气道上皮整合素β4可诱导性剔除小鼠模型，我们确证了胚胎期气道上皮整合素β4表达缺陷致小鼠支气管肺发育不良，表现为气道上皮细胞成熟障碍，多层细胞重叠；肺泡隔减少，肺泡面积增大；气道上皮细胞纤毛减少、倒伏；肺组织中与发育相关的分子TTF1、SHH、BMP4及11β-HSD1表达明显减低，肺表面活性蛋白SPB、SPA和肺泡Ⅱ型标志物（SPC）表达明显减少。气道上皮整合素β4沉默的小鼠肺功能受损，表现为粘液纤毛清除功能受损；自发炎症反应增强；LPS诱导的肺部炎性细胞浸润加重并激活M1型巨噬细胞。同时，气道上皮整合素β4表达缺陷的肺组织出现一定程度的ECM异常重构及硬度增加；不同发育阶段的肺组织mRNA转录组测序可见细胞运动、细胞轴动力、细胞外基质相关基因表达差异明显。在细胞水平证实整合素β4沉默增加人气道上皮细胞损伤修复迁移过程中的细胞骨架刚度，抑制黏着斑形成与成熟，破坏细胞与基底的稳定粘附，使上皮细胞迁移速度变慢，整合素β4沉默组细胞在硬基底上的骨架稳定性明显降低。以上结果提示胚胎期气道上皮细胞整合素β4表达缺陷将影响上皮-基质信号传递及胎肺对宫内外环境变化的抵抗力，从而导致肺发育不良并与肺发育不良相关疾病的发生发展密切相关。本研究将拓展粘附分子与肺发育内在联系的认识，寻求以整合素β4作为肺发育先天性疾病防治新靶点的可能，同时也有助于加深对成人获得性肺疾病的发生发展的理解。