PCV2感染对猪淋巴细胞NF-κB信号通路及炎性细胞因子表达的动态影响  

Dynamic effects of PCV2 on NF-κB signaling pathway and inflammatory cytokine expression in lymphocytes of piglets in vitro

作　　者：董林[1,2] 王艳萍 唐娜[1,2] 徐倩倩[1] 苗立中 王金良[1,2] 沈志强[1,2] DONG Lin;WANG Yanping;TANG Na;XU Qianqian;MIAO Lizhong;WANG Jinliang;SHEN Zhiqiang(Binzhou Animal Science and Veterinary Medicine Institude,Binzhou,Shandong 256600,China;Shandong Lvdu Bio-Sciences&Technology Co.Ltd.,Binzhou,Shandong 256600,China)

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东绿都生物科技有限公司,山东滨州256600

出　　处：《中国兽医学报》2022年第6期1115-1121,共7页Chinese Journal of Veterinary Science

基　　金：山东省重大科技创新工程资助项目(2019JZZY020606);山东省自然科学基金资助项目(ZR2017MC056)。

摘　　要：研究PCV2感染猪淋巴细胞对核因子NF-κB信号通路的影响,探索其在NF-κB信号通路中调控炎性细胞因子生成作用机制。选取10头28日龄PCV2抗原、抗体双阴性仔猪,无菌采集、分离脾淋巴细胞,制成单细胞悬液,分PCV2感染组和对照组进行体外培养。感染组PCV2病毒添加剂量250μL/100 mL细胞悬液,对照组加入同体积细胞培养液,培养后0,6,12,24,48 h收集淋巴细胞和上清液。共聚焦电镜检测NF-κB核移位,Western blot检测NF-κB/P65核蛋白、磷酸化抑制性κBα蛋白含量,EMSA检测NF-κB与DNA的结合率,ELISA试剂盒检测上清液中IL-1β、TNF-α、IL-6、IL-10、IL-17和TGF-β1的含量。结果显示,NF-κB/P65蛋白在淋巴细胞核中移位程度随着PCV2感染时间增加而增强,6 h后试验组显著高于对照组(P<0.05)。NF-κB与DNA的结合率与PCV2感染时间呈现线性增长,PCV2感染后6 h试验组显著高于对照组(P<0.05),48 h有所降低,但相对对照组差异不显著(P>0.05)。淋巴细胞质中p-IκBα蛋白表达在PCV2感染后24 h达到峰值,且在整个检测期间相对对照组差异极显著(P<0.01)。PCV2感染组IL-1β和IL-6含量在6,12 h与对照组差异不显著(P>0.05),24 h有所升高,48 h感染组IL-1β和IL-6含量显著高于对照组(P<0.05)。IL-17和TNF-α随着PCV2感染时间延长含量不断增多,6 h显著高于对照组(P<0.05),24 h达到峰值,48 h极显著高于对照组(P<0.01)。IL-10含量呈现明显的先升高后降低的趋势,PCV2感染组6 h开始升高,12 h显著高于对照组(P<0.05),24 h开始降低,48 h与对照组基本一致。TGF-β1含量呈现先降低后升高的趋势,感染组6~12 h含量显著低于对照组(P<0.05),24~48 h明显升高。结果表明,PCV2通过磷酸化降解途径显著激活淋巴细胞中NF-κB信号通路,使其核移位和DNA结合率显著增强,调控炎性细胞因子的表达。通过IL-10和TGF-β1的升高或降低调控机体在感染早期表现免疫抑制,后期出现全身炎症反应的病The effects of PCV2 on nuclear factor NF-κB signaling and inflammatory cytokines were studied to explore its role in regulating of pro-inflanmmatory cytokine production and NF-κB signaling pathways in lyphocytes of PCV2 infected piglets.Ten piglets of 28 day-old were chosen as the experiment animal that were PCV2 antigen and antibody double negative.The cells were isolated aseptically from spleens with pinhead of injectors and were made single cell suspension,which was divided into PCV2 infection group and control group for in vitro culture.The lymphocytes of the infected group was incubated with PCV2 at a dose of 250 μL/100 mL cell suspension,and the control group was incubated with the equal volume cell culture medium.The PCV2-inoculated and mock-inoculated lymphocytes and supernatant at 0,6,12,24 and 48 h post-inoculation were collected,respectively.Nuclear translocation of NF-κB was detected by confocal laser scanning microscope.NF-κB/P65 nuclear protein and phosphorylation inhibitory protein were detected by Western blot and the binding rate of NF-κB to DNA was detected by electrophoretic mobility sfit assays(EMSA).The contents of IL-1β,TNF-α,IL-6,IL-10,IL-17 and TGF-β1 in the cells supernatant were detected by ELISA kit.The degree of translocation of NF-κB/P65 protein in the lymphocyte nucleus increased with the increase of PCV2 infection time,which was significantly higher in the PCV2 infected group than the control group after 6 h post-inoculation(P<0.05).The binding degree of NF-κB to DNA and the inculated time of PCV2 showed a linear increase that was significantly higher in the PCV2 infected group than the control group(P<0.05),which was decreased at 48 h post-inoculation and the difference was not significant compared with the control group(P<0.05).The expression contents of p-IκBα protein in the lymph cytoplasm peaked 24 h post-inoculation,which was significantly different from the control group during the whole detection period(P<0.01).The contents of IL-1β and IL-6 in the PCV2 infecte

关 键 词：PCV2 淋巴细胞 NF-ΚB 信号通路 细胞因子 

分 类 号：S852.4[农业科学—基础兽医学]