Dok-1参与嗜碱性粒细胞脱颗粒信号转导机制的研究

Food-induced allergic reactions are mainly attributed to IgE-mediated mechanisms that binding of food allergens by specific IgE on basophils or mast cells initiates a complex series of signaling cascades followed by the release of inflammatory mediators causing a variety of symptoms. Many researchers have described the downstream of tyrosine kinase (Dok)-1 as a negative regulator of immunoreceptor signaling, activated by inhibitory receptors such as FcγRIIB in mast cells. However, we found that Dok-1 could also modulate signaling processes initiated by clustering an activating receptor — i.e. FcεRI. Dok-1 was found to undergo a marked and time-dependent tyrosine phosphorylation upon FcεRI stimulation and phosphorylated Dok-1 may establish a vital role in regulating the FcεRI-mediated mast cell degranulation. However, it is still unclear how the signals diverge at Dok-1 to produce these different effects and how Dok-1-mediated pathways regulate FcεRI-mediated mast cell functions. In this study, we examine the signal transduction mechanism that activate Dok-1 using isolated human umbilical cord blood-derived basophils by magnetic separation and established RBL-2H3 cell model. Furthermore, we also investigate the molecular mechanism for Dok-1 in FcεRI-mediated mast cell degranulation by generation of cells overexpressing Dok-1 variants, using several techniques including flow cytometry, confocal microscopy and iTRAQ proteomics. The results will provide scientific basis for elucidating the molecular mechanism of food allergy and detecting a potential target in suppressing allergen-mediated inflammatory processes.

食物过敏主要是由嗜碱性粒细胞表面FcεRI结合的特异性IgE与相应过敏原相互作用引发一系列信号级联反应后诱发细胞脱颗粒而导致的。很多科学家认为嗜碱性粒细胞酪氨酸激酶下游分子(Dok)-1通过激活FcγRIIB抑制受体来抑制FcεRI 诱发的脱颗粒信号反应。我们前期研究发现Dok-1可以直接被FcεRI受体激活，进而调节细胞脱颗粒反应，但是Dok-1在FcεRI激活后是如何被磷酸化的以及磷酸化的Dok-1是通过什么信号转导途径调节细胞脱颗粒反应尚不清楚。本项目拟分离纯化人脐带血嗜碱性粒细胞，结合前期研究已建立的RBL-2H3细胞免疫学模型，探索FcεRI交联后激活Dok-1的信号转导机制；构建Dok-1稳定超表达细胞系，结合流式细胞术、激光共聚焦以及iTRAQ蛋白组学等技术，探究激活的Dok-1参与的细胞脱颗粒信号的转导机制。研究结果为阐明食物过敏过程发敏阶段的免疫学机制提供科学依据。

食物过敏主要是由嗜碱性粒细胞表面FcεRI结合的特异性IgE与相应过敏原相互作用引发一系列信号级联反应后诱发细胞脱颗粒而导致的。很多科学家认为嗜碱性粒细胞酪氨酸激酶下游分子(Dok)-1通过激活FcγRIIB抑制受体来抑制FcεRI诱发的脱颗粒信号反应。前期研究发现Dok-1可以直接被FcεRI受体激活，进而调节细胞脱颗粒反应，但是Dok-1在FcεRI激活后是如何被磷酸化的以及磷酸化的Dok-1是通过什么信号转导途径调节细胞脱颗粒反应尚不清楚。本项目拟分离纯化人脐带血嗜碱性粒细胞，结合前期研究已建立RBL-2H3细胞免疫学模型，探索FcεRI交联后激活Dok-1的信号转导机制；构建Dok-1稳定超表达细胞系，结合流式细胞术、激光共聚焦以及iTRAQ蛋白组学等技术，探究激活的Dok-1参与的细胞脱颗粒信号的转导机制。结果发现抗原刺激FcεRI激活后，Dok-1通过与PI3K的产物PI(3,4,5)P3相结合，从胞质区域转移至质膜区域，进而被质膜区域的Src家族激酶（如Lyn，Fyn）磷酸化；磷酸化的Dok-1抑制了由TRPC1通道控制的细胞外Ca2+内流反应，将Nck招募至质膜区域，通过抑制Ca2+依赖的F-肌动蛋白微丝解聚负调节细胞脱颗粒反应。研究结果为阐明食物过敏过程发敏阶段的免疫学机制提供科学依据。