猪圆环病毒2型诱导PK-15细胞产生IFN-β的信号通路研究

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

【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(P<0.01),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(P<0.01),TLR9的表达量在48、72 h显著升高(P<0.01);TLR3和TLR9下游的接头蛋白MyD88和TRIF的mRNA 含量在48 h显著升高(P<0.01),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(P<0.01),RIG-1的mRNA 含量在72 h显著升高(P<0.01),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(P<0.01),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(P<0.01),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(P>0.05),TBK1/IKKε抑制剂组的IFN-β的mRNA 含量与PCV2组相比明显下降(P<0.05),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。

【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 0.5 μmol BX795, 5 μmol BAY 11-7082 and 0.5 μmol BX795＋5 μmol 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 72 h 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 48 h and 72 h（P0.01）, 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 48 h and 72 h（P0.01）, the mRNA levels of TLR3 increased obviously at 48 h（P0.01）, and the mRNA levels of TRIF and MyD88 which were adaptor proteins of TLR9 and TLR3 were also up-regulated at 48 h（P0.01）, 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 72 h（P0.01）, the expression levels of IRF3,MAVS, Sting which were adaptor proteins of RIG-1 and MDA-5 significantly increased at 48 h（P0.01）, indicating that IRF3 signal pathway mediated by RLRs is activated by PCV2. The mRNA levels of DAI were higher at 48 h and 72 h（P0.01）, 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（P0.01）, 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.