摘要
为研究猪圆环病毒2型(Porcine circovirus type 2,PCV2)感染的猪肺泡巨噬细胞(Porcine alveolar macrophages,PAMs)分泌Ⅰ型干扰素信号通路,以PCV2病毒感染PAMs为研究对象,采用酶联免疫吸附测定(Enzyme-linked immunosorbent assay,ELISA)、实时荧光定量PCR和Western blotting,分析PCV2感染对PAMsⅠ型干扰素的诱导、cGAS/STING信号通路相关基因mRNA和蛋白表达的影响,并应用靶向cGAS和STING特异性si RNA、抑制剂BX795和BAY 11-7082,解析cGAS、STING、TBK1和NF-κB/P65在PAMs生成Ⅰ型干扰素中的作用。结果显示,PAMs感染PCV2病毒48 h后Ⅰ型干扰素的表达量显著升高(P<0.05),cGAS mRNA的表达量在感染48 h和72 h后显著升高(P<0.01),STING mRNA表达量在PCV2感染72 h后显著上升(P<0.01),TBK1mRNA、IRF3 mRNA感染48 h后显著升高(P<0.01)。PCV2能够显著升高PAMs胞浆STING、TBK1和IRF3蛋白含量,降低胞浆NF-κB/p65的含量,促进NF-κB/p65和IRF3入核。敲低PAMs中cGAS或STING表达水平后,PCV2感染PAMs 48 h后,Ⅰ型干扰素的表达水平显著下降(P<0.01);BAX795抑制TBK1后,PCV2感染PAMs48 hⅠ型干扰素的表达水平显著下降(P<0.01),BAY 11-7082抑制NF-κB/P65表达后,PCV2感染PAMs 48 hⅠ型干扰素的表达量与PCV2组相比无显著性差异(P>0.05)。结果表明,PAMs感染PCV2后通过cGAS/STING/TBK1/IRF3信号通路诱导Ⅰ型干扰素分泌。
In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages(PAMs) infected with porcine circovirus type 2(PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type Ⅰ interferon(IFN-Ⅰ) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-Ⅰ increased significantly at 48 h after infection with PCV2(P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection(P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection(P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection(P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-Ⅰ was significantly decreased after PCV2 infection for 48 h(P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-Ⅰ in BX795-treated group was significantly reduced than that of the PCV2 group(P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-Ⅰ secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.
作者
陈洪博
李凤
赖文琰
方宇豪
江铭涌
段滇宁
杨小燕
Hongbo Chen;Feng Li;Wenyan Lai;Yuhao Fang;Mingyong Jiang;Dianning Duan;Xiaoyan Yang(College of Life Sciences,Longyan University,Longyan 364012,Fujian,China;Fujian Provincial Key Laboratory for the Prevention and Control of Animal Infectious Diseases and Biotechnology,Longyan 364012,Fujian,China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2021年第9期3201-3210,共10页
Chinese Journal of Biotechnology
基金
福建省科技重大专项(No.2019NZ09005)
中央引导地方科技发展专项项目(No.2019L3011)
福建省自然科学基金(Nos.2018J01461,2019J01803)
大学生创新创业训练项目(No.202011312054)资助。
