囊泡分选蛋白VPS33B在血小板α颗粒形成中的机制研究

血小板颗粒内容物的释放参与多种重要的生理病理过程，内容物能否正确运载取决于颗粒的正确形成，α颗粒作为主要运载体，目前对其形成过程及机制了解甚少。VPS33B异常可导致α颗粒缺失。我们用免疫沉淀结合蛋白质谱筛选出多种可能存在相互作用的蛋白，包括VIPAR、NBEAL2和Rab蛋白家族等，但它们的具体作用机制尚不明确。本研究拟分别从细胞和动物水平阐述VPS33B蛋白在α颗粒形成中的作用机制：通过免疫沉淀和蛋白印迹方法验证与VPS33B相互作用蛋白并结合荧光共聚焦显微镜确定这些蛋白胞内定位；采用shRNA技术特异性敲除某种蛋白，观察其他相关蛋白的胞内定位；构建巨核系/血小板VPS33B条件敲除小鼠，通过系列血小板功能试验和诱导止血发生实验在体评价血小板α-颗粒缺失对凝血过程的影响。结果有望揭示血小板α-颗粒形成的细胞生物学机制，为阐述血小板α-颗粒异常疾病发病机制提供理论基础。

Platelet is the major effector cells during thrombosis process, which releases kinds of granule contents involved in a variety of important pathophysiological processes. α-granule, as the primary granule in platelet, carries more than 200 kinds of proteins. The progress of granule biogenesis is the important step to determine whether the contents can be properly carried. But little is known about the mechanisms and formation process of platelet α-granule. Mutations in VPS33B lead to platelet α-granule deficiency. In our previous experiments, some proteins interacting with VPS33B were screened out using immunoprecipitation in combination with MS method, including VIPAR, NBEAL2 and Rab protein family, but the specific mechanism of interaction of these proteins is unclear. In this study we will expound the formation mechnism of VPS33B protein in platelet α-granule with animal model and in cells level (MEG01 cells and HEK293T cells), respectively: using immunoprecipitation and western blot methods to validate the proteins interacting with VPS33B; using fluorescent confocal microscopy to determine intracellular localization of these proteins; using the shRNA technology to specificly knock out a certain protein to observe the intracellular localization of other related proteins; megakaryocytic / platelet VPS33B conditional knockout mouse model was adopted to evaluate the influence of platelet α-granule deficiency during haemostasis and thrombostasis process in vivo, through a series of platelet function tests and thrombus model analysis. All these results are contributed to elucidate the mechanism of platelet α-granule formation and will provide a theoretical basis for elaborating the pathogenesis of platelet α-granule deficiency.