气道上皮细胞凋亡是慢性气道炎性疾病重要的病理生理现象。研究者在已结题NSFC项目中发现，分泌型CLCA1刺激钙激活氯离子通道TMEM16A高表达的气道上皮细胞后，细胞凋亡率显著增加，提示CLCA1/TMEM16A和气道上皮细胞凋亡有关。基于氯离子通道和细胞容积调控的关系，我们推测TMEM16A的过度活化及其继发的凋亡相关容积减少可能是引发凋亡的关键。本项目拟在体内外模型中研究CLCA1/TMEM16A和气道上皮细胞凋亡的关系，探讨CLCA1减弱TMEM16A与脂肪筏区主要蛋白caveolin1的结合力，使TMEM16A转位至非脂肪筏区激活的机制，以及激活后细胞内渗透压、细胞体积调节能力、牛磺酸外流量和凋亡相关基因表达的变化。预期结果将阐明CLCA1活化TMEM16A，进而触发凋亡相关容积减少、导致气道上皮细胞凋亡的现象和机制，为气道上皮细胞凋亡和慢性气道炎症的治疗探索出新的治疗靶点。

Apoptosis of bronchial epithelial cells (BEC) plays an important role in the pathogenesis of chronic airway inflammatory diseases. It was found in the last NSFC project（81200028） study that secreted Chloride Channel Accessory 1 (CLCA1) could significantly increase the rate of apoptosis in the bronchial epithelial cells transfected with transmembrane member 16A (TMEM16A). It indicates that apoptosis of BEC is associated with CLCA1/TMEM16A and the speculated mechanism is that apoptosis is induced by an excessive activation of TMEM16A and apoptotic volume decrease (AVD) induced by TMEM16A. In the present study, we will look into the role of TMEM16A in the apoptosis of BEC in vivo and in vitro by up-regulating or down-regulating CLCA1 or TMEM16A expression. We will also study the possible processes, that CLCA1 activates TMEM16A due to lessen the strength of interaction between TMEM16A and Caveolin-1 which is the main protein of lipid rafts and relocate TMEM16A to non-raft fractions by Co-Immunoprecipitation (Co-IP), GST-pulldown assays and Sucrose centrifugation assays. A further study will be conducted to explore if over-activation of TMEM16A could induce AVD directly. We will detect intracellular osmotic pressure, cell volume regulation and taurine efflux after TMEM16A activation. We seek to characterizes the role of CLCA1/TMEM16A in inducing apoptosis and regulating airway inflammatory-state , demonstrate the mechanism of CACL1 inducing TMEM16A activation and the mechanism of TMEM16A inducing AVD, providing a new therapeutic target for epithelial cell apoptosis and chronic airway inflammatory diseases.

气道上皮细胞凋亡是慢性气道炎性疾病重要的病理生理现象。研究者在已结题NSFC项目中发现，分泌型CLCA1刺激钙激活氯离子通道TMEM16A高表达的气道上皮细胞后，细胞凋亡率显著增加，提示CLCA1/TMEM16A和气道上皮细胞凋亡有关，本项目在体外HBE16细胞模型中研究CLCA1/TMEM16A和气道上皮细胞凋亡的关系，探讨CLCA1激活后细胞内渗透压、细胞体积调节能力和凋亡相关基因表达的变化。因此，我们从以下方面进行了研究：第一，探索CLCA1/TMEM16A相互作用引起上皮细胞凋亡的研究；第二，研究CLCA1/TMEM16A介导凋亡相关体积减少的机制；第三，研究 CLCA1/TMEM16A与下游凋亡基因表达和活性的关系。实验结果如下：第一， 将HBE16 细胞经转染 Ad-TMEM16A 24-48h 后，采用PCR和Western Blot实验方法测定TMEM16A mRNA和蛋白表达水平，其结果较对照组显著升高，转染Ad-siRNA-TMEM16A后，TMEM16A mRNA 和蛋白表达水平较对照组显著降低，然而Ad-siRNA-Scramble腺病毒载体后，TMEM16A mRNA 和蛋白基本不变。第二，在CLCA1 200ng/ml、刺激24 小时后，细胞凋亡及炎性因子的分泌随TMEM16A转染表达增高而上升，降低而减少。第三，置于低渗环境中，凋亡体积减少（AVD）随TMEM16A转染表达增高而下降，降低而上升。第四，在CLCA1 200ng/ml、刺激24 小时时，caspase-3， bax mRNA和蛋白随TMEM16A转染表达增高而上升，降低而减少。bcl-2 mRNA和蛋白随TMEM16A转染表达增高而降低，上升而减少。综上所述，我们的结果表明通过CLCA1活化TMEM16A，然后调控下游凋亡基因表达和活性进而触发凋亡相关容积减少、导致气道上皮细胞凋亡。