拴链因子Exocyst复合物在慢性气道炎症中的促黏液分泌效应

Mucin hypersecretion is a notable clinical and pathological feature in chronic inflammatory diseases of the airway. The process is involved five steps: coating, translocation, tethering, docking and fusion, and exocytosis. However, the mechanisms of the polar motion of secretory vesicles and the tethering between the membranes of vesicle and plasma membrane are unclear. On the basis of intracellar location and cellular dynamics, the tethering factor,termed Exocyst complex, may play an important role in those processes. In this study, the normal human bronchial epithelial cells were exposed to the powerful human neutrophil elastase, then the activation of phosphorylated Sec3 protein is examed by Western blotting, the expression and localization of Exocyst complex are deserved by freeze-etching electron microscopy, and the difference of secreted mucin between the upper and underneath compartments of Transwell is measured by ELISA. To test the effects and key sites of Sec3 protein on Exocyst complex activation and mucin expression, we constructed the mutants at the possible sites by TALEN experiment. To test the interaction between Sec3 and Rab-GTPase, GST pulldown and immunoprecipitation are selected. The aim of the study is to further measure the mechanism of mucin hypersecretion and provide direction for its treatment.

气道黏液高分泌是慢性气道炎性疾病的重要临床病理特征，该分泌过程包括黏蛋白的包被、转运、拴链、膜之间的对接融合和出胞五个步骤，然而目前对于分泌颗粒的极向移动及其与出胞活跃位点的拴链结构基础尚不明确。根据细胞动力学原理，该效应可能与拴链因子Exocyst复合物的作用有关。本研究以中性粒细胞弹性蛋白酶为刺激因素，观察Sec3的磷酸化、Exocyst的表达和定位以及极向培养细胞黏蛋白分泌的相位差异，明确Exocyst及Sec3在黏蛋白极性分泌过程中的作用；通过构建Sec3定点突变体，明确其在Exocyst活化过程中的作用及关键作用位点；进而构建Rab TAL干扰体，免疫荧光双重染色观察Sec3和Rab的细胞定位，GST-pulldown 分析及免疫共沉淀法分析Sec3和Rab的相互作用，明确Rab在Exocyst介导的黏蛋白极性分泌过程中的调控作用，以期最终对气道黏蛋白的出胞分泌机制进行深入诠释。

在本项目研究周期内，课题组成员通过细胞实验以及动物模型研究，以中性粒细胞弹性蛋白酶为刺激因素，通过观察Exocyst的表达和定位、Sec3的磷酸化、以及极向培养细胞黏蛋白分泌的相位差异，已明确Exocyst及Sec3在黏蛋白极性分泌过程中的作用；通过构建Sec3定点突变体，已明确其在Exocyst活化过程中的影响及关键作用位点；加之通过构建Rab TAL干扰体，免疫荧光双重染色Sec3和Rab的细胞定位，GST-pulldown 分析及免疫共沉淀法分析Sec3和Rab的相互作用，明确了Rab在Exocyst介导的黏蛋白极性分泌过程中的调控作用；证实了Sec3蛋白和Rab-GTP酶（Rho/Cdc42）之间的相互作用可影响Exocyst复合物各亚基的招募、上膜和组装，从而影响Exocyst复合物在拴链过程中的组装、循环和解离过程的推测。在项目进行的过程中，本课题组成员本着严谨求实的科研态度，较圆满地完成了该基金项目的预期目标，目前已发表26篇论著，包括13篇SCI论文，且尚存有较多研究成果行将发表。本项目的研究进一步完善了炎性情况下黏蛋白包被、转运、拴链、膜之间对接融合和出胞这一连续而复杂的细胞动力学过程，为炎性气道黏液高分泌的防治提供全新的启示。