气道皮层肌动蛋白结合肽Cortactin介导粘蛋白出胞的分子链接机制

Mucus hypersecretion is the major pathological features of chronic airway inflammatory disease.This process is an increase of mucin exocytosis.The increase of exocytosis of secretory granules which packages concentrated mucin was related to the mediation of the cortical cytoskeleton and the upregulation of exocytosis sites.However, the mechanism of mediated secretory granules through a very dense cell cortex pole to the plasma membrane and increases the exocytosis sites is not clear.Based on the consideration of intracellular localization and functional characteristics of the cortical actin binding peptide (Cortactin),We think that the process should be related to the role of the cytoskeleton and cortactin and the impact on the cell cortex membrane fluidity.In this study,the human airway epithelial cells will be cultured in vitro,and stimulated by abnormally elevated shear that imitates airway obstruction.Then,we observe the expression and localization of Cortactin. Meanwhile,we will construct mutant gene expression vector of Cortactin, so as to clear its role in mucin secretion process and the key action site.Immunofluorescence double staining will be used to observe the ellular localization of cortactin and MARCKS.With GST pull down method and immunoprecipitation, the interaction of cortactin and MARCKS will also be analyzed. The corresponding disrupters for the important signaling molecules during this process will be given to analyzed the key role, and using Fluorescence recovery after photobleaching to detect cell membrane fluidity.We expect to provide guidance for the therapy and prevention of airway mucus hypersecretion.

黏液高分泌是慢性气道炎性疾病的重要病理特征，系由粘蛋白出胞活动的增加导致的，此行为的增加与皮层内细胞骨架的介导及出胞位点的上调有关，然而介导分泌颗粒穿越致密皮层极向移动并增加出胞位点的机制尚不明确。基于目前对皮层肌动蛋白结合肽Cortactin胞内定位和功能特性的认识进步，该过程应关乎Cortactin和细胞骨架的相互作用及对膜流动性的影响。本研究通过培养人气道上皮细胞，以气道阻塞时升高的剪切力为刺激因素，观察Cortactin的表达及定位；构建Cortactin突变表达载体，明确其在粘蛋白分泌中的作用及关键位点；免疫荧光双染色观察Cortactin和胞吐关键调控因子MARCKS定位的关系、GST pulldown 法和免疫共沉淀分析两者的相互作用；给予相应的干扰制剂对该过程的重要信号分子进行分析，并采用荧光漂白恢复技术观察细胞膜流动性的相应演变，以期诠释粘蛋白出胞的部分下游分子链接机制。

黏液高分泌是慢性气道炎性疾病的重要病理特征，系由粘蛋白出胞活动的增加导致的，此行为的增加与皮层内细胞骨架的介导及出胞位点的上调有关，然而介导分泌颗粒穿越致密皮层极向移动并增加出胞位点的机制尚不明确。基于目前对皮层肌动蛋白结合肽Cortactin胞内定位和功能特性的认识进步，该过程应关乎Cortactin和细胞骨架的相互作用及对膜流动性的影响。本研究通过体外培养人气道上皮细胞，以气道阻塞时升高的剪切力、中性粒细胞弹力蛋白酶为刺激因素，观察Cortactin、F-actin的表达及定位；构建Cortactin不同磷酸化位点突变表达载体，明确其在粘蛋白分泌中的作用及关键位点；免疫荧光双染色观察Cortactin和胞吐关键调控因子MARCKS定位的关系、免疫共沉淀分析Cortactin与ACK1可能存在的相互作用。同时给予相应的干扰siRNA对该过程的重要信号分子进行分析，以期诠释粘蛋白出胞的部分下游分子链接机制。结果显示， Cortactin Tyr421、Tyr470位点突变载体降低F-actin的聚合，减少j剪应力所致的MUC5AC分泌，在黏蛋白MUC5AC胞外分泌过程中起更重要的作用。中性粒细胞弹力蛋白酶刺激后MACRCKs与Cortactin 共表达增加，F-actin的聚合增强，MUC5AC分泌增加，同时，ACK1的表达也有增强，免疫共沉淀检测结果提示Cortactin和ACK1存在相互作用。MARCKs siRNA转染后，磷酸化ACK1及磷酸化Cortactin蛋白表达明显减少，ACK1 siRNA转染后, Cortactin磷酸化水平降低。CO-IP显示Cortactin 与ACK1相结合。综上所述，结果表明， Cortactin与MARCKs参与了气道黏液分泌过程，Cortactin Tyr421、Tyr470位点起着较为重要的作用。MARCKs通过增加ACK1活性，促使Cortactin磷酸化和F-actin极化和易位，共同参与MUC5AC分泌。