C-RAF激酶及其结合蛋白对肥大细胞功能影响作用机理研究

Epidemiologic survey have shown that the prevalence of allergic diseases in the world population is as high as 15-30%, it becomes a global health problem endangering human health, and is regarded as one of three major diseases by WHO (World Health Organization) with emphasis on their prevention and treatment in this century. C-Raf kinase is the key knot protein of C-Raf-MEK-MAPK signaling pathway which is at the heart of signaling networks. Through interacting with numerous its binding proteins, C-Raf kinase efficiently regulates the functions of dendritic cells, Th2 cells, and B cells in the process of allergic reactions. In this project, in order to find out the effect of C-Raf kinase on mast cells functions and the interaction of C-Raf-binding proteins with C-Raf, we will study the influence of C-Raf with different expression levels on mast cells activation, the expression and release of inflammatory cytokines and determine the change of C-Raf kinase activity and the spectrum of C-Raf-binding proteins in different states of mast cells (resting and activation states). In our previous study, it has been shown that different kinds of allergens can efficiently activate immune cells involved in allergies such as mast cells, but the mechanism is unclear (Molecular & Cellular Proteomics 2013). In this project, we will define the mechanism by which C-Raf kinase and its binding proteins regulate mast cells functions in the process of allergic reaction, and provide theoretical basis and potential targets for prevention and treatment of allergic diseases.

流行病学调查显示过敏性疾病的发病率高达15-30%，已成为严重影响人类健康的全球性疾病，被世界卫生组织列为本世纪重点防治的三大疾病之一。作为细胞信号通路主干枢纽蛋白的C-Raf激酶，通过与其结合蛋白相互作用，调控免疫细胞在过敏反应发生过程中的作用，但尚不了解C-Raf对过敏反应最直接的效应细胞肥大细胞功能的影响。本项目拟通过干扰肥大细胞C-Raf的表达来研究其对肥大细胞脱颗粒及炎症因子生成与释放的作用，同时观察不同状态下肥大细胞中C-Raf的活性及C-Raf结合蛋白谱的变化，来研究C-Raf及其结合蛋白对肥大细胞功能的影响。我们前期研究结果表明过敏原蛋白可以有效地活化肥大细胞，但其具体作用机制尚不清楚（Molecular & Cellular Proteomics 2013）。通过该研究阐述C-Raf及其结合蛋白对肥大细胞功能调控的作用机制，为过敏反应性疾病的诊治提供理论依据和潜在的靶标。

过敏性疾病是严重影响人类健康的全球性疾病。作为细胞信号网路主干通路的枢纽蛋白C-Raf激酶，通过与其结合蛋白相互作用，调控免疫细胞在过敏反应发生过程中的作用，但尚不了解C-Raf激酶对过敏反应发生最直接效应细胞肥大细胞功能的影响。.本研究利用互作蛋白质组学的方法，分别构建了C-Raf基因敲除和C-Raf过表达稳定肥大细胞系。系统分析了C-Raf过表达肥大细胞在活化和静息状态下C-Raf结合蛋白谱的变化。通过比较肥大细胞不同状态下的C-Raf结合蛋白谱，我们选取了3种可能与肥大细胞活化相关的C-Raf结合蛋白进行了深入研究。探讨这些结合蛋白对肥大细胞释放炎症因子、脱颗粒、肥大细胞迁移及肥大细胞活化相关信号通路等的影响。找到了2个（Rap1b和L-Plastin）对肥大细胞功能有重要影响的C-Raf结合蛋白，为诊断和治疗过敏反应提供了新的潜在靶标。目前共发表标注本项目资助的SCI论文7篇，其中影响因子>5的2篇，核心期刊论文8篇。申请发明专利3项，获授权专利1项，出版学术专著1部，培养博士生1名（在读），硕士生3名。.总之，该项目完成了任务书规定的任务，构建了肥大细胞在静息状态和活化状态下的C-Raf激酶结合蛋白网络，发现了2个潜在的用于诊断和治疗过敏反应的新靶点。同时也为进一步深入研究肥大细胞活化发生的分子机制做了物质和人才上的储备。