猪圆环病毒2型诱导PK-15细胞产生IFN-β的信号通路研究

【目的】研究猪圆环病毒2型(porcine circovirus type2,PCV2)感染PK-15细胞后调控β-干扰素(interferon-β,IFN-β)生成的信号通路,为猪圆环病毒病的发生机理和防治提供理论基础。【方法】将PK-15细胞随机分成5组:对照组、PCV2组、BX795(TBK1/IKKε抑制剂)组、BAY 11-7082(NF-κB抑制剂)组和BX795+BAY11-7082(混合抑制剂)组。BX795组、BAY 11-7082组、BX795+BAY 11-7082组分别用0.5μmol BX795、5μmol BAY11-7082、0.5μmol BX795+5μmol BAY 11-7082预先处理1 h,然后感染PCV2。于感染后3、12、24、48和72 h收集细胞,提取RNA。用荧光定量PCR检测IFN-β、模式识别受体(TLR3、TLR9、RIG-1、MDA-5、DAI)、接头蛋白(TRIF、MyD88、Sting、MAVS、IRF3)的mRNA 含量。【结果】PCV2感染PK-15细胞后,IFN-β的mRNA 含量在48、72 h显著升高(P0.01),表明PCV2感染可以诱导PK-15细胞生成IFN-β;TLR3的mRNA 含量在48 h显著升高(P0.01),TLR9的表达量在48、72 h显著升高(P0.01);TLR3和TLR9下游的接头蛋白MyD88和TRIF的mRNA 含量在48 h显著升高(P0.01),表明PCV2激活了Toll样受体介导的NF-κB信号途径;MDA-5的mRNA 含量在48、72 h显著升高(P0.01),RIG-1的mRNA 含量在72 h显著升高(P0.01),MDA-5和RIG-1下游的接头蛋白MAVS、Sting、IRF3的mRNA 含量在48 h显著升高(P0.01),表明PCV2激活了RIG-1样受体介导的IRF3信号途径;DAI的mRNA 含量在48、72 h显著升高(P0.01),表明PCV2亦激活了DNA模式识别受体介导的IRF3信号途径;分别用BX795和BAY 11-7082抑制IRF3和NF-κB介导的信号通路,结果显示NF-κB抑制剂组的IFN-β的mRNA 含量与PCV2组相比无显著性差异(P0.05),TBK1/IKKε抑制剂组的IFN-β的mRNA 含量与PCV2组相比明显下降(P0.05),表明PCV2诱导IFN-β的产生主要由IRF3信号途径调控。【结论】PCV2感染可以诱导PK-15细胞中IFN-β表达量的上调,其上调主要与IRF3信号通路有关。

Signal Pathway of Interferon-β Induced by Porcine Circovirus Type 2 in PK-15 Cells

【Objective】 The objective of this study is to investigate the mechanism of IFN-β production induced by PCV2 in PK-15 cells and provide a theoretical foundation for the pathogenesis and prevention of porcine circovirus disease.【Method】 The PK-15 cells were divided into five groups randomly: Control group, PCV2 group, BX795 (inhibitor of TBK1/IKKε) group, and BAY11-7082 (inhibitor of NF-κB) group, BX795+BAY 11-7082(mixed inhibitors) group. BX795 group, BAY 11-7082 group and BX795+BAY 11-7082 group were pretreated with 0.5µmol BX795, 5µmol BAY 11-7082 and 0.5µmol BX795+5µmol BAY 11-7082 for one hour, respectively. Then the PK-15 cells were infected with PCV2 for 1 h except the control group. The cells were collected at 3, 12, 24, 48 and 72h after infection. The RNA was extracted from the five groups and then reverse-transcribed into cDNA. The mRNA levels of IFN-β, pattern recognition receptors (TLR3, TLR9, RIG-1, MDA-5 and DAI) and adaptor proteins (TRIF, MyD88, Sting, MAVS and IRF3) were detected by real time PCR.【Result】The mRNA levels of IFN-β in PCV2-infected groups were significantly higher than the control at 48h and 72h (P＜0.01), indicating that IFN-β can be induced by PCV2 in PK-15 cells. The mRNA levels of TLR9 in PCV2-infected groups were higher than the control at 48h and 72h (P＜0.01), the mRNA levels of TLR3 increased obviously at 48h (P＜0.01), and the mRNA levels of TRIF and MyD88 which were adaptor proteins of TLR9 and TLR3 were also up-regulated at 48h (P＜0.01), demonstrating that NF-κB signal pathway mediated by TLRs is activated by PCV2. The mRNA levels of MDA-5 and RIG-1 were significantly higher than the control at 72h (P＜0.01), the expression levels of IRF3, MAVS, Sting which were adaptor proteins of RIG-1 and MDA-5 significantly increased at 48h (P＜0.01), indicating that IRF3 signal pathway mediated by RLRs is activated by PCV2. The mRNA levels of DAI were higher at 48h and 72h (P＜0.01), also indicating that DNA recognition receptor is involved in the production of IFN-β. BX795 and BAY 11-7082 inhibitors were used to inhibit the signal pathway mediated by IRF3 and NF-κB, the result showed that the mRNA levels of IFN-β in BX795-treated group was obviously lower than the control group (P＜0.01), while no significant difference was found between BAY 11-7028-treated group and the control group, demonstrating that the up-regulation of IFN-β induced by PCV2 is mainly relative to the IRF3 signal pathway. 【Conclusion】The PCV2 could induce production of IFN-β in PK-15 cells and IRF3 signal pathway played an important role during this process.