Anti-β2GPⅠ/β2GPⅠ引起的血小板活化和血栓形成中p38MAPK信号转导通路作用的研究

Thrombotic diseases have become an important disease of the threat to human health and life, its pathogenesis and treatment is one of the focus and the hotspots for contemporary medical research. Pletelet activation is the key to thrombosis, and the p38MAPK signal transduction pathway in pletelet activation can not be ignored. Preliminary findings of our research group was that platelet activation levels and platelet aggregation rate increased in the anti-β2GPI positive patients, and phosphorylation p38MAPK expressed strongly . However, there is no report at home and abroad so far whether anti-β2GPⅠ/β2GPⅠ combined with the platelet receptor to induce platelet activation and aggregation by p38MAPK signal transduction pathway and the role of platelet receptor(apoER2' and GPⅠbα) affect the level of the p38MAPK phosphorylation . Therefore, our research group intend to use the dimer β2GPⅠ as an inducer to prove the role of p38MAPK signal transduction pathway in anti-β2GPI/β2GPI induced platelet activation and thrombosis through in vitro and in vivo experiments. We will use the technology of chemical block, gene knockout, flow cytometry, immunoblotting, laser scanning confocal microscopy, OCT and so on. We will observe from various angles platelet receptors affect the level of the P38MAPK phosphorylation in dimer β2GPⅠ induced platelet activation and thrombosis , further improve the mechanism of anti-β2GPI/β2GPI induced platelet activation and thrombosis, and provide new ideas for the treatment of autoantibody-related thrombotic diseases.

血栓性疾病严重威胁了人类健康，其发病机制与治疗是医学研究的重点之一。血小板活化是血栓形成的关键，其中的p38MAPK信号转导通路作用更是不容忽视。本课题组前期研究发现anti-β2GPⅠ阳性患者的血小板活化程度增高、磷酸化p38MAPK表达增强。但是anti-β2GPⅠ/β2GPⅠ是否是结合血小板受体（apoER2'和GPⅠbα）通过p38MAPK信号转导通路使血小板活化和聚集，迄今国内外尚无报道。因此，本课题组拟用二聚化β2GPⅠ作为诱导剂，通过体外及体内实验，利用化学阻断、基因敲除、流式细胞术、免疫印迹、超声血流仪、LSCM、OCT等技术，全方位研究在二聚化β2GPⅠ引发血小板活化和血栓形成中p38MAPK的磷酸化水平，以及apoER2'和GPⅠbα对其的影响，从而进一步完善anti-β2GPⅠ/β2GPⅠ引起血小板活化和血栓形成的机制，为自身抗体相关的血栓性疾病的治疗提供新思路。

抗β2GPⅠ抗体与其靶抗原β2GPⅠ结合形成的anti-β2GPⅠ/β2GPⅠ复合物可以通过干扰β2GPⅠ抗凝或与细胞膜表面受体结合促使细胞活化，参与机体血栓形成。血小板活化是血栓形成的关键因素，其胞内p38 MAPK可以介导多种细胞内反应，在血小板相关血栓形成中发挥重要作用，又因其具有调节细胞迁移、增殖、分化、凋亡等作用，常作为肿瘤或炎症性疾病的治疗靶点。本课题旨在探讨anti-β2GPⅠ/β2GPⅠ复合物通过p38MAPK调控血小板活化、促使血栓形成的作用机制。.通过体外实验，以p38MAPK抑制剂，GPⅠbα和/或apoER2’拮抗剂作为干预因素，探讨p38MAPK及各受体在anti-β2GPⅠ/β2GPⅠ复合物诱导血小板活化和血栓形成中的相互作用。通过体内实验，建立野生/基因敲除小鼠颈总动脉血栓模型，揭示p38MAPK、GPⅠbα和apoER2’在anti-β2GPⅠ/β2GPⅠ复合物诱导血小板活化、聚集、分泌和血栓形成中的作用。.体外实验显示，anti-β2GPⅠ/β2GPⅠ复合物可促使血小板p38MAPK磷酸化水平升高，CD62p和PAC-1表达增加，微颗粒释放增加，血小板聚集率升高；GPⅠba和/或apoER2’拮抗剂可使该现象得到显著抑制，且单独/联合抑制作用无显著差异。体内实验显示，anti-β2GPⅠ/β2GPⅠ复合物注射后小鼠血管闭塞时间缩短，相同时间血管内血栓形成面积增加，小鼠外周血血小板p38MAPK活化水平升高，CD62p和PAC-1表达增加，血小板聚集率升高；注射p38MAPK抑制剂后，小鼠血管闭塞时间明显延长，相同时间血管内血栓形成面积减少，小鼠外周血血小板p38MAPK活化水平降低，CD62p和PAC-1表达减少，血小板聚集率降低。.本研究清楚的证实了anti-β2GPⅠ/β2GPⅠ复合物可以通过与细胞膜表面受体GPⅠbα和apoER2’同时结合激活p38MAPK信号转导通路活化血小板，促使血小板聚集和分泌，并提示了这一机制参与了anti-β2GPⅠ/β2GPⅠ复合物导致的血栓事件的发生和发展，有望为揭示anti-β2GPⅠ/β2GPⅠ复合物促血栓形成的发病机制及自身抗体相关血栓形成的预防和治疗提供新的思路与靶点。

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