NCS-1调控Ca2+/CamKⅡ信号通路在支气管哮喘肥大细胞脱颗粒中的作用机制

Mast cells (MC) are pivotal trigger and effective cells in the development of chronic inflammation of asthma. Challanged MC can lead to asthma attack by synthesizing and then releasing histamine and other inflammatory mediators in the form of degranulation upon stimulation. However, the mechanism of this process is unclear. Preliminary work from our research group has already confirmed that MC shows a rich expression of neuronal calcium sensor protein(NCS-1); the NCS-1 expression is significantly increased in sensitized MC in asthma; NCS-1 binding with Ca2+ can activate Ca2+/CaMKII signal pathway and promote the release of intracellular calcium and an increase in the intracellular concentration of calcium ions; NCS-1 expression on the membrane of vesicles are significantly enhanced. Thus we speculate NCS-1 can trigger asthma attack by activating the Ca2+/CaMKII and increasing the level of histamine and other inflammatory mediators which released from MC secretory vesicles regulated positively by [Ca2+]i. On the basis of established model of asthmatic rats, the mast cells were isolated, purified, cultured and then used siRNA interference to suppress or transfected with virus vector to upregulate the expression of NCS-1. Furthermore, according to the different levels of NCS-1 expression in MC, the effect of NCS-1 on Ca2+/CaMK‖signal pathway were investigated to explore the pathogenesis of asthma identify by the release of histamine- - the marker of degranulation at the molecular level.It also provide the theortical evidence for finding new theraputic taget point in asthma.

肥大细胞(MC)是哮喘慢性炎症发生发展过程中关键的触发细胞和效应细胞，致敏的MC大量合成组胺等炎性介质，以脱颗粒形式释放导致哮喘发作，但其机制不甚明了。本课题组前期工作已证实：MC富表达神经钙传感蛋白NCS-1；哮喘致敏的MC中NCS-1表达显著增加，其活化态可激活Ca2+/CaMKⅡ信号通路，促进细胞内钙库释放，增加细胞内钙离子浓度([Ca2+]i)；同时囊泡质膜表达显著增强。由此我们推测NCS-1通过激活 Ca2+/CaMKⅡ信号通路正向调控MC钙触发的分泌囊泡脱颗粒，释放组胺等导致哮喘发作。在建立哮喘大鼠模型的基础上分离、纯化、浓缩和培养MC；采用siRNA干涉抑制或病毒载体细胞转染改变NCS-1表达，进一步检测MC中不同NCS-1水平对 Ca2+/CaMKⅡ信号通路及脱颗粒标志物组胺释放量的影响，从分子水平上深入地探讨哮喘的发病机制，为临床寻找哮喘干预的靶点提供理论依据。

哮喘的本质是一种气道慢性炎症，体内许多炎性细胞参与其炎症的发生和发展过程，嗜酸粒细胞和T淋巴细胞起着重要的调节作用, 特别是肥大细胞（Mast Cell, MC）是哮喘慢性炎症发生和发展过程中关键的触发细胞和效应细胞。肥大细胞是一种典型的免疫-内分泌细胞，可在激活后具有大量合成和释放炎性介质的功能和特性，导致哮喘的发作，但其发生机制报道较少。本课题在哮喘大鼠模型的基础上，成功的利用磁珠分选法和不连续的Percoll密度梯度溶液二种方法分离、纯化和浓缩肺组织中肥大细胞，并进行了有效的细胞培养,为siRNA抑制和慢性病毒感染过表达提供了实验保障。同时为从基因和蛋白水平探讨NCS-1通过调控Ca2+/CaMKII水平，从而控制炎性释放的机理成为可能。本实验相关结果表明，MC富表达神经钙传感蛋白 NCS-1；和正常组比较,哮喘组NCS-1的表达显著增加；NCS-1的表达受siRNA及SFV-NCS-1融合蛋白的双向调节，进一步的研究表明,采用 siRNA 干涉抑制或病毒载体细胞转染改变 NCS-1 表达，会引起CaMKⅡ活性、MC内Ca2+浓度及脱颗粒标志物组胺释放量的相应变化。首次证实了神经钙传感蛋白NCS-1可与Ca2+结合活化，通过激活 Ca2+/CaMKⅡ信号通路正向调控 MC 钙触发的分泌囊泡脱颗粒，释放组胺等导致哮喘发作；证实和明了了神经钙传感蛋白 NCS-1参与了哮喘炎症的合成和释放的崭新的分子细胞生物学途径，为寻找可能阻断或抑制哮喘炎症反应的药物提供了新思路和理论依据。本项目培养和训练了多名博硕研究生，使他们不仅达到了很好的实验技能的训练，同时也培养他们进行基础研究的思维，使他们熟悉如何进行材料的分析、整理和论文的撰写等系列的培养过程；已撰写和本课题密切的论文3篇，已发表相关SCI论文6篇，中文核心期刊1篇；本课题解决了有关肥大细胞基础研究中长期存在分离纯化和培养困难的问题，为今后其他实验室进行类似研究提供技术支持和借鉴；同时把NCS-1应用到免疫炎性细胞的研究当中，突破了神经调控蛋白应用到外周细胞的研究的创新。