PKCs-p66Shc信号通路介导的气道上皮细胞线粒体损伤在COPD发病中的作用机制研究

Our previous study has demonstrated that p66Shc adapter protein related to mitochondria may be involved in the progression of COPD, through modulating mitochondrial function of airway epithelial cell. In order to further elucidate the role of PKCs-p66Shc signaling pathway in the pathogenesis of COPD, this project has been designed. First, primary cultured airway epithelial cell will be stimulated with cigarette smoke extract. The role of p66Shc and its interaction with FKBP38 in the course of mitochondrial damage of airway epithelial cell will be studied using gene transfection, RT-PCR, Wb, co-immunoprecipitation, GST Pull-down and so on. Second, the regulative effect of PKCs on p66Shc will be explored during the mitochondrial damage of airway epithelia cell by gene silencing and PKCs inhibitor intervention. Last, a rat model of COPD will be induced by cigarette smoke, and the rats will be treated with PKCs inhibitors. The effect of PKCs-p66Shc signaling pathway on mitochondrial damage of airway epithelium will be studied in those rats. The purpose of this project is to provide scientific basis for further studying the role of mitochondria in the pathogenesis of COPD and targeted therapy for COPD.

我们前期研究发现，与线粒体相关的衔接蛋白p66Shc可能通过影响气道上皮细胞线粒体功能，参与慢性阻塞性肺疾病（COPD）发病过程。为进一步阐明p66Shc及其上游调节分子PKCs在COPD发病中的作用，本项目拟首先用香烟烟雾提取物刺激原代培养的大鼠气道上皮细胞，采用基因转染、RT-PCR、Wb、免疫共沉淀、GST Pull-down等技术，明确p66Shc对气道上皮细胞线粒体损伤的分子机制以及与线粒体膜蛋白FKBP38相互作用对线粒体损伤的影响；其次通过基因沉默和PKCs抑制剂干预，确定p66Shc对气道上皮细胞线粒体损伤的调控依赖PKCs信号分子；最后香烟烟雾刺激建立COPD大鼠模型，同步应用PKCs抑制剂下调PKCs活性，明确PKCs-p66Shc信号通路在COPD大鼠气道上皮细胞线粒体损伤中的作用。本课题的实施与完成，为从线粒体角度探索COPD的发病机制及靶向治疗提供重要科学依据。

慢性阻塞性肺疾病（COPD）是呼吸系统常见疾病，在我国40岁以上人群中的患病率为13.7%，总体患病人数约9990万，然而该病的发病机制迄今仍不清楚。本项目拟从气道上皮细胞线粒体角度阐明PKCs-p66Shc信号通路在COPD发病中的作用机制。. 首先，观察p66Shc在COPD大鼠肺上皮中的表达水平。采用香烟烟熏联合气管内注射脂多糖方法建立COPD大鼠模型，HE染色表明模型组大鼠存在小气道周围炎症、肺气肿等COPD病理学特征；应用透射电子显微镜发现COPD大鼠肺泡上皮线粒体存在严重损伤；免疫组化研究显示，p66Shc及其磷酸化水平在COPD大鼠肺上皮细胞中显著升高。其次，研究香烟烟雾提取物（CSE）对p66Shc表达及上皮细胞的影响。随着CSE干预浓度的升高，MTT结果显示细胞活性逐渐降低，real-time PCR和western-blot结果表明细胞内p66Shc的mRNA和蛋白表达水平逐渐上升，线粒体内p66Shc含量显著增加；7.5%CSE干预气道上皮细胞0-48h后，细胞活性逐渐降低，细胞内p66Shc的mRNA和蛋白表达水平呈时间依赖性升高，同时增加p66Shc的线粒体转位；p66Shc siRNA预处理，可显著减轻CSE对气道上皮细胞及其线粒体的损伤。最后，探讨PKC抑制剂预处理对CSE诱导的气道上皮细胞损伤的影响。PKCβ抑制剂（LY333531）和PKCδ抑制剂（rottlerin）预处理气道上皮细胞，可显著抑制CSE诱导的p66Shc表达水平的增加及其线粒体转位，减轻CSE诱导的细胞和线粒体损伤。. 本研究表明，PKCs-p66Shc信号通路介导的气道上皮细胞线粒体损伤参与COPD发病，为从线粒体角度探索COPD发病机制及靶向治疗提供重要科学依据。