非典型CSFV云南株调控血管内皮细胞ASC介导的炎症信号通路机制

Classical swine fever (CSF) is one of the serious world recognized disease harm to the development of pig industry, which is attended by the governments and the scientific community. In recent years, the clinical features of classical swine fever take the direction of the atypical CSF, the pigs infected by atypical CSFV does not show typical symptoms of hemorrhage in various tissues and organs, but the CSFV nucleic acid can be detected in the spleen, mandibular lymph and tonsil. In the study of last National Natural Science Foundation of China, we found that the inflammatory cytokine transcription levels were significant difference between atypical swine fever virus and Shimen strain. Therefore, depth exploration for the mechanism of inflammation-related signaling pathways affected by atypical CSFV Yunan strain will be explorated. After the vascular endothelial cells infected by atypical CSFV Yunan strain, ubiquitination level, nuclear-cytoplasmic shuttling, activation of the inflammasome of ASC, which was the key adapter protein in the upstream of the inflammatory signaling pathways, were detected. Then, the secretion of cytokines, mRNA levels, protein expression and nuclear translocation controlled by NF-κB, which was the central regulator of intracellular inflammatory mediators signal, were detected too. By these studies, the mechanism of the inflammatory signaling pathway impacted by the atypical CSFV Yunnan strain will be Clarified, and then provide important theoretical support for the control of atypical CSF in Yunnan province.

猪瘟是世界公认的严重危害养猪业发展的疾病之一，受到各国政府和科学界的重视。近年来我国猪瘟的发病特点向非典型性方向发展，非典型猪瘟病毒感染猪不表现为各个组织器官广泛性出血等典型的猪瘟症状，却可以在脾脏、下颌淋巴和扁桃体中可检验出猪瘟病毒核酸。在前一项国家自然科学基金的研究中，我们发现非典型猪瘟病毒对机体炎症相关细胞因子转录水平的影响和猪瘟病毒石门株相比差异显著，因此本项目对非典型猪瘟病毒云南株影响机体炎症相关信号通路的机制进行深入探索。将非典型猪瘟病毒云南株感染猪血管内皮细胞后，通过检测炎症信号通路上游中的关键接头蛋白ASC的泛素化水平、核质穿梭、炎症小体的激活，再检测下游细胞内炎性介质信号的中心调节因子NF-κB控制的相关细胞因子的分泌mRNA转录水平、蛋白表达量以及核转位等，阐明非典型猪瘟病毒云南株影响炎症信号通路的机制，这对云南非典型猪瘟的防控提供重要的理论支撑。

本课题对非典型CSFV云南株对炎症信号通路上游ASC以及下游NF-κB的影响进行研究。通过建立体外感染模型，采用MTT法和CCK-8法检发现，接种CSFV 24h后细胞活性显著下降，TCID50法检测发现，20h-48h是病毒的高速繁殖期；通过ELISA的方法检测炎性因子IL-6、IL-1β、TNF-α、TGF-β，发现不同时间点内的细胞因子浓度均出现上升；通过荧光定量PCR检测NFKBIA、IKBKB、CHUK、RelA基因，发现CSFV感染ECs 后，IKBKB和RelA的mRNA转录水平明显升高，CHUK的转录水平有上升但不明显，只有NFKBIA的表达降低。Western blot检测结果表明，炎性相关蛋白p65、IKKα、IKKβ、p-IKKα、IκBα、p-IκBα均在总蛋白中表达，在胞浆中IKKα、IKKβ、p-IKKα、IκBα、p-IκBα表达，IKKα的表达在所有变化中均不明显。在胞核中p65、p-p65表达。构建出真核表达质粒pcDNA3.1-Flag-ASC（Flag-ASC）、pcDNA3.0-HA-Ub（HA-Ub）、pCAGGS-Myc-TRAF3（Myc-TRAF3）和pCAGGS-Myc-MAVS（Myc-MAVS）；将Flag-ASC和HA-Ub表达质粒共转染血管内皮细胞，接种CSFV，免疫共沉淀检测CSFV感染血管内皮细胞后ASC的泛素化水平，发现CSFV感染血管内皮细胞4h后，ASC出现明显的泛素化修饰；敲低细胞内MAVS和TRAF3的表达发现MAVS和TRAF3参与CSFV感染后ASC的泛素化过程；通过ASC核质穿梭的研究发现，RNA病毒感染后ASC存在核质穿梭，并且TRAF3参与RNA病毒感染后ASC的核质穿梭过程；通过MAVS与TRAF3对炎症小体激活的研究发现，MAVS和TRAF3均能再细胞体内与ASC相互作用，且MAVS对ASC的调控可能依赖于TRAF3；CSFV感染的ECs细胞中 pro-caspase-1 发生明显切割，p10 含量增加，IL-1β水平升高，Caspase-1活性增强，进一步说明了CSFV感染血管内皮细胞后MAVS与TRAF3能促进炎症小体的激活。这对云南非典型猪瘟的防控提供重要的理论支撑。