整合素β1在肺纤维化去细胞支架促上皮-间质转化中的作用及机制研究

Idiopathic pulmonary fibrosis (IPF) is a kind of senile disease, the pathogenesis remains unclear. The studies show that myofibroblasts are the major effector in the fibrous lesions. Epithelial mesenchymal transition (EMT) of type II alveolar epithelial cells (AT II) is one of the main sources of myofibroblasts. Therefore, further study on EMT is of great significance for pulmonary fibrosis. It was found that changes of the microenvironment of extracellular matrix (ECM) can induce the change of cell stress, which is an important inducement of cell phenotype regulation. In the previous study, we found that the content of integrin beta 1 protein in the pulmonary fibrosis acellular scaffold (ECM) was significantly higher than that in normal lung acellular scaffold (normal ECM). Moreover, we found that AT II gradually showed the characteristics of interstitial cells after be implanted into 3D pulmonary fibrosis ECM. So we speculate that it may be related to the increased expression of integrin beta 1, but the detailed mechanism is not clear. In this study, to clarify the role and mechanism of ECM in EMT，the AT II will be co-cultured with 3D lung acellular scaffold and the phenotype transformation of AT II will be observed, the expression and phosphorylation level of the related genes and proteins in mechanochemical pathway mediated by integrin beta 1 will be detected. It will lay the experimental foundation for the subsequent search for new therapeutic targets of pulmonary fibrosis from integrin beta 1 mediated mechanochemical pathway.

特发性肺纤维化(IPF)是一种老年病，发病机制不明，研究表明肌成纤维细胞是肺纤维化病灶的主要效应细胞，而肺泡Ⅱ型上皮细胞（ATⅡ)经上皮间质转化（EMT)是其主要来源之一。因此深入研究EMT意义重大。研究发现细胞外基质（ECM）微环境结构变化可致细胞受力因素改变，后者是细胞表型调控的重要诱因。课题组前期研究发现纤维化肺脱细胞支架（纤维化ECM）中整合素β1蛋白含量较正常肺脱细胞支架（正常ECM）明显增多。同时将ATⅡ植入3D肺纤维化ECM，可见部分ATⅡ逐渐呈现出“间质细胞特性”，推测可能与整合素β1表达增多有关，但具体机制尚不明确。本研究将ATⅡ与3D纤维化肺脱细胞支架共培养，观察ATⅡ表型转化情况，并检测整合素β1介导的机械生化通路相关基因蛋白表达及其磷酸化水平。阐明肺纤维化ECM在ATⅡ发生EMT中的作用与机制，为后续从整合素β1介导的机械化学通路寻找新的治疗靶点奠定实验基础。

特发性肺纤维化(IPF)发病机制不明，研究表明肌成纤维细胞是肺纤维化病灶的主要效应细胞，而肺泡II型上皮细胞(ATII)经上皮间质转化(EMT)是其主要来源之一 。因此深入研究EMT意义重大。研究发现细胞外基质(ECM)微环境结构变化可致细胞受力因素改变，后者是细胞表型调控的重要诱因。本项目从在体动物实验检测整合素 β1 介导的 FAK-Src-p130Cas 机械生化信号传导通路及其下游 FAK-Ras-MAPK 通路在肺纤维化中的表达变化。并通过制备正常及肺纤维化去细胞生物支架，构建3D肺去细胞支架体外细胞培养体系，探讨正常与纤维化肺去细胞支架对II肺肺泡上皮细胞的活化作用及机制。结果发现，在体实验中，与正常对照组相比，免疫组化法、WB、组织免疫荧光和荧光定量PCR检测结果显示博来霉素诱导大鼠肺纤维化后肺组织中整合素β1，FAK，Src，P130cas，ERK1/2，MEK1/2，P38及其磷酸化蛋白及相关基因的表达水平明显升高（P＜0.05），而整合素β1阻断剂ATN-161干预后可缓解大鼠肺纤维化，下调了纤维化肺组织中的整合素β1，FAK，Src，P130cas，ERK1/2，MEK1/2，P38及其磷酸化蛋白及相关基因的表达水平（P＜0.05）。离体细胞实验显示3D肺纤维化脱细胞生物支架可促进ATII发生EMT， WB、细胞免疫荧光和荧光定量PCR检测通路蛋白（整合素β1，FAK，Src，P130cas，ERK1/2，MEK1/2，P38及其磷酸化蛋白）及相关基因的表达水平，其中3D肺纤维化脱细胞生物支架组的整合素β1，FAK，Src，P130cas，ERK1/2，MEK1/2，P38及其磷酸化蛋白及相关基因的表达均高于对照组（P＜0.05），而整合素β1阻断剂ATN-161可下调以上蛋白及基因的表达并抑制EMT（P＜0.05）。本项目明确了整合素β1介导的FAK-Scr-p130Cas机械生化信号传导通路及其下游FAK-Ras-MAPK通路在肺纤维化中起着重要作用，为后续寻找新的肺纤维化临床干预治疗靶点奠定实验基础，并且本项目发现整合素β1阻滞剂ATN-161可以抑制博来霉素诱导的大鼠肺纤维化以及抑制3D肺纤维化脱细胞支架促进 II 型肺泡上皮细胞 EMT的进展过程，可能成为将来肺纤维干预治疗的潜在药物之一。