ANCA诱导的ROS在调控中性粒细胞凋亡∕NETosis转换中的作用机制

Neutrophil has a pivatol role in anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis(AAV). ANCA can induce the generation of reactive oxygen species (ROS)of neutrophil, which is thought to be the key point of the pathogenitis of AAV. In 2004, a novel type of neutrophil death, neutrophil extracellular trap(NET) cell death, which is also named NETosis, was reported. Upon activation, neutrophils release NETs and then die. NETs are composed of chromatin decorated with granular proteins such as myeloperoxidase, proteinase 3 et al. NETs provide a high local concentration of antimicrobial molecules that kill microbes effectively. NETs are abundant at inflammatory sites, and were shown to be relevant in AAV. ANCA has been reported to be able to induce NETosis. Our previous study has demonstrated that the generation of NETs depended on the level of ROS induced by ANCA. Low-level of ROS could only result in the acceleration of apoptosis of neutrophil, while high-level of ROS could induce the NETosis. So we thought that the ROS should be pivotal to the conversion between apoptosis and NETosis. In the further study, we plan to investigate the role of ROS in the conversion between apoptosis and NETosis further. Apoptosis will be detected with flow cytometry and NETosis will be detected with the measurement of DNA concentration. The signal pathway involved in the induction of apoptosis and NETosis of neutrophil by ANCA and the relationship between apoptosis and NETosis of neutrophil in AAV will be investigated. We will also study the possibility to enhanve the apoptosis of neutrophils by blocking the NETosis.So the further study will help us to verify the pathogenesis of AAV and to find effective therapies of the disease.

抗中性粒细胞胞浆抗体（ANCA）是ANCA相关小血管炎（AAV）的标志抗体，可以诱导中性粒细胞产生活性氧（ROS)。NETosis发现于2004年，指中性粒细胞释放出由胞内DNA及各种酶组成的胞外网(NETs），进而发生死亡的现象。NETs对机体具有很强的损伤性，ANCA可以诱导NETs形成。课题组前期研究表明，ANCA是否诱导NETosis依赖于ROS的水平，低水平ROS仅诱导细胞凋亡，高水平ROS则可诱导NETosis。故ROS可能在中性粒细胞从凋亡到NETosis的转换中起关键作用。本研究拟在此基础上采用流式细胞术测定凋亡，利用定量测定DNA检测NETs生成等方法，进一步研究ANCA刺激中性粒细胞时ROS在调控凋亡∕NETosis中的作用及信号通路，明确中性粒细胞凋亡与NETosis之间的关系，并抑制NETosis使细胞向凋亡转化，为从新的角度探究AAV的发病机制及治疗提供理论基础。

中性粒细胞胞外诱捕网（NETs）在ANCA相关性血管炎（AAV）的发病中具有重要作用，我们对NETs在AAV血栓形成中的作用机制进行了研究，并重点对NETs与活动期ANCA相关血管炎重要的血清标志物C反应蛋白（CRP）共同作用机制进行了研究。从正常人外周血分离中性粒细胞，用含有ANCA的IgG刺激细胞产生NETs并将细胞包被在固相载体，利用含CRP的富血小板血浆进行灌注。结果发现netting中性粒细胞可在剪切力作用下使血小板沉积并活化，并且使五聚体CRP（pCRP）沉积在活化血小板上并转化为单体CRP（mCRP），mCRP可引起更多的血小板活化，mCRP还可增强灌注实验中血浆D-二聚体的形成，同时增强血小板分泌炎症因子HMGB-1。说明剪切力作用下血小板可沉积在ANCA刺激的netting中性粒细胞上并发生激活，同时循环中的pCRP沉积在激活的血小板上并解离为mCRP，从而进一步激活血小板及凝血系统，提示NETs促使了ANCA相关血管炎患者血栓事件的发生，而高水平的CRP可能通过转化为mCRP而进一步加强这种作用。ANCA结合到中性粒细胞引起后者的活化是AAV的核心致病机制，为进一步探讨ANCA对中性粒细胞NETs生成的关系，我们对血液中重要的负性炎症标志物--白蛋白（Albumin，ALB）在ANCA刺激中性粒细胞活化过程中的所发挥的作用进行了研究。我们收集了74名抗MPO抗体阳性AAV患者临床资料，发现血清ALB与CRP呈显著负相关性（R=-0.536, P<0.001）。为了证明AAV中ALB对炎症的抑制作用是通过抑制ANCA刺激中性粒细胞NETs产生而实现的，我们研究了在不同浓度的ALB的条件下ANCA刺激中性粒细胞产生NETs能力的变化，发现从AAV患者获取的中性粒细胞较正常人对ANCA刺激NETs生成的作用更加敏感，随着ALB浓度的增加，ANCA刺激中性粒细胞NETs的产生受到抑制，在ALB浓度为20g/L和40g/L时ANCA刺激中性粒细胞产生的ROS水平具有明显差异（P<0.05）。我们同时发现中性粒细胞产生NETs水平也有显著性差异（P<0.05），同时发现ANCA的亲和力对此过程具有影响，亲和力强的ANCA受ALB封闭作用的影响较小，而亲和力低的ANCA受ALB封闭作用的影响更大，进一步证明了NETs在AAV的发病机制中具有重要作用。