SFTS病毒介导巨噬细胞吞噬及损伤血小板的分子机制

Severe fever with thrombocytopenia syndrome (SFTS) caused by infection of severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging disease. Severe thrombocytopenia is the clinical feature of SFTS. It has been found that macrophages can phagocytose SFTSV-adherred platelets, but its molecular mechanism is unknown. According to the published data and our previous research results, we presume that superficial proteins of SFTSV may link platelets with macrophages to activate macrophages to phagocytose the platelets as well as damage the phagocytosed platelets through phagolysosome formation. In this study, we will use the typical experimental methods such as confocal microscopy, flow cytometry and Western Blot assay to determine the direct combination of viral envelope protein Gn with cellular integrins though RGD motif and the indirect combination of viral envelope protein Gc with cellular integrins though the proteins in extracellular matrix to cause the viral adherence to platelets and macrophages. The viral adherence activate the integrin-coupled FAK and its downstream PI3K signaling pathways of macrophages to cause the aggregation of microfilament and microtubule as well as rearrangement of cystoskeleton that stimulates the macrophages to phagocytose the virus and virus-adhered platelets. The fusion of phagocytotic vesicles and lysosomes in the macrophages to form phagolysosomes that causes the damage and degradation of the virus and virus-adhered platelets. The results of this study will illuminate the molecular mechanism of thrombocytopenia in SFTS patients during SFTSV infection which is a novel finding with high medical significance.

发热伴血小板减少综合征（SFTS）病毒是新发传染病病原，血小板显著减少是患者临床主要特征之一，但其分子机制不明。根据文献及前期研究结果，巨噬细胞能吞噬病毒粘附的血小板，我们推测SFTSV表面蛋白能易化血小板与巨噬细胞间连接，进而激活巨噬细胞吞噬血小板形成吞噬溶酶体导致血小板损伤。本项目拟采用激光共聚焦显微镜、流式细胞术和Western Blot等方法，探讨SFTSV包膜蛋白Gn和Gc能否通过RGD基序直接与整合素结合或经胞外基质分子间接与整合素结合后黏附血小板和巨噬细胞，分析巨噬细胞中整合素偶联的FAK及其下游PI3K信号通路，以及微丝和微管聚集及细胞骨架重排在引发巨噬细胞吞噬病毒及其黏附的血小板中的作用，吞噬泡与溶酶体融合形成吞噬溶酶体而导致病毒及其黏附的血小板破坏和降解，初步阐明SFTSV感染后患者血小板数量明显减少的分子机制，具有显著临床意义。

发热伴血小板减少综合症（SFTS）病毒是新发传染病病原，中国已将SFTS列为重点监控的新发传染病之一。血小板减少症是发热伴血小板减少综合征病毒 （SFTSV）感染患者的常见并发症，但其致病机制仍不明确。既往对SFTSV感染机制的研究主要集中于免疫系统（免疫器官、免疫细胞、免疫分子等），极少量研究涉及血小板。鉴于此，本项目从血小板抗SFTSV感染免疫的独特视角出发，突破新病原感染宿主研究的传统思路，揭示了血小板参与免疫应答是导致SFTS患者产生血小板减少综合症的关键机制之一。研究团队通过细胞培养技术获得病毒分离株，采用透射电镜、扫描电镜、荧光逆转录PCR等手段，首次发现血小板能够迅速吞噬血液中的SFTSV，证明血小板是血管内抵抗病原体的第一道防线。通过结构生物学和血小板黏附抑制实验证明了SFTSV能够直接与血小板膜上的糖蛋白GPVI结合，从而介导病毒黏附血小板并导致血小板活化。随后研究团队从分子、细胞和动物模型等不同水平，采用激光共聚焦、流式细胞术等手段，阐明血小板活化使得血管内皮细胞P-选择素表达上调、血小板铺展、血小板回缩、血小板凋亡和血栓形成等一系列反应，从而导致患者血小板计数减少的机制。同时，进一步研究证明了血小板能够促进巨噬细胞的吞噬作用，但SFTSV在巨噬细胞中的复制加剧血小板消耗，是SFTSV感染引起血小板持续性下降和其他并发症的主要原因。本项目揭示了血小板参与机体抗SFTSV感染的独特功能，对探究SFTSV感染导致血小板减少的致病机制提供关键的技术支撑，对SFTS的防治提供了理论依据和潜在的分子靶标，对未来血小板抗感染免疫的研究提供了新的思路和策略，具有显著临床意义。