氯离子在气道上皮应激中的早期信号分子作用研究

In the previous study, we observed a decrease of chloride ions flux and Cl-1 accumulation in various kind of cells including bronchial epithelial cells (BEC), insulted by different stress stimuli including temperature stress, acid-base stress, antigen insulting and oxidant, etc. However, different dynamics of Cl-1 accumulation could be induced by different kind of stimulus. Therefore, a hypothesis can be speculated that chloridion is possibly the early signal molecules during airway epithelium stress. The differences among mechanisms of channel gating for chloride ions flux results in the differences of dynamics of Cl-1 accumulation, which activate those Cl-1 sensed-proteins and its signal pathway, consequently induce different stress responses on airway epithelial cells including proliferation and wound repair, immune response, and inflammatory adhesive activation, etc. To certificate the hypothesis, this study aim at to definite Cl-1 as the early signal molecules during airway epithelium stress, and make sure the relation of the dynamics of Cl-1 accumulation—Cl-1 signal pathway—stress response type of cells. The main research content includes: ①the dynamics of Cl-1 accumulation in BEC stimulated by different stress, foe example, either transient accumulation or persistent accumulation, and different gating mechanism of channels including CFTR and CACC; ②the change of cellular function with Cl-1 accumulation in BEC, observing cells proliferation and speed of wound repair, uptake and presentation of antigen by BEC, adhesion with inflammatory cells such as PMB,EOS and monocytes, secretion of inflammatory cytokines, and definite the relationship between dynamics of Cl-1 accumulation and immune type or inflammation type; ③combine whole wide genome siRNA data bank and protein data bank (PDB), selecting those proteins which both involve in above stress response and has the domain to bind Cl-1 ,and for further verification. This research will discover the early mechanism in airway epithelium stress, and provide reasons to explain the different response type and end results of airway epithelium after being stressed.

申请者观察到人支气管上皮细胞（BEC）等多种细胞在不同应激刺激下出现胞内氯离子浓度增加及其动力学方式的差异，推测氯离子可能作为气道上皮应激的早期信号分子。氯离子流动的通道门控机制差异导致氯离子浓度动力学方式差异，进而激活不同的氯离子敏感蛋白及其信号通路，产生不同的应激反应定式，包括增殖修复、炎症和免疫激活定式。为确定氯离子动力学方式-氯离子信号通路-细胞应激功能定式的对应关系，拟研究：①应激刺激下BEC内氯离子浓度增加的动力学方式，以及相对应的通道机制；②胞内氯离子浓度增加伴随的细胞功能改变，包括细胞增殖和修复速率、抗原摄取和提呈、炎性细胞粘附激活等，建立氯离子动力学方式与免疫或炎症定式的对应关系；③筛选并验证具有氯离子结合域且参与上述细胞功能的蛋白质。该研究将确定氯离子作为新的气道上皮应激早期信号分子，阐明气道上皮应激反应的早期启动机制，为解释气道上皮应激后的不同反应定式及转归提供依据。

本项目根据前期课题研究线索，人支气管上皮细胞等多种细胞在不同应激刺激下出现胞内氯离子浓度增加及其动力学方式的差异，推测氯离子可能作为气道上皮应激的早期信号分子。氯离子流动的通道门控机制差异导致氯离子浓度动力学方式差异，进而激活不同的氯离子敏感蛋白及其信号通路，产生应激反应定式，包括增殖修复和炎症等。本项目确定氯离子动力学方式-氯离子信号通路-细胞应激功能定式的对应关系，研究结果如下：①利用尼日利亚菌素（nigericin）和三丁基氢化锡（tributyltin）双离子载体配制不同的氯离子浓度钳制液，用氯离子敏感性荧光探针MQAE，结合激光共聚显微镜定量分析细胞内氯离子浓度，确证细胞内氯离子浓度在各种应激刺激下应答性增高，氯离子通道CFTR、 TMEM16A和CLIC4 表达及氯电流响应刺激变化；②用氯离子通道抑制剂与特定[Cl-]i钳制液，建立胞内高氯离子浓度气道上皮细胞模型，观察[Cl-]i增高对气道上皮细胞损伤修复的影响以及力学性质的变化，与静息状态下的氯离子浓度相比，[Cl-]i一定程度的增高可激活RhoA/ROCK/LIMK信号通路，使细胞骨架发生重组，降低细胞刚度，促进细胞迁移，加快损伤修复速度；③细胞内氯离子通道CLIC4在LPS诱导的上皮细胞炎性反应中的表达下降，伴细胞内氯离子浓度升高，参与上皮细胞炎性反应的调节，其作用与对上皮细胞氯离子转运的调控有关；④氯离子通道抑制剂或将细胞内氯离子钳制在高浓度增强了细胞内FAK的磷酸化。细胞内高氯通过激活FAK，进而激活PI3K/AKT信号通路，促进人支气管上皮细胞细胞的增殖和损伤修复能力。本研究明确了氯离子作为气道上皮应激早期信号分子，通过氯离子信号通路调节气道上皮细胞的增殖和创伤愈合能力的新机制，为解释气道上皮应激反应的早期启动机制及转归提供依据。