免疫复合物对猪繁殖与呼吸综合征病毒诱导的PAM细胞因子转录的影响

Effects of Immune Complex on the Transcription of Cytokines in PAM Cells Induced by Porcine Reproductive and Respiratory Syndrome Virus

本试验通过研究免疫复合物影响猪繁殖与呼吸综合征病毒(PRRSV)在巨噬细胞内增殖的因素,来进一步探讨免疫复合物经FcγR介导的PRRSV感染机制。本研究将含200TCID50PRRSV病毒液与等体积的终浓度为1.30mg·mL-1的猪IgG-兔抗猪IgG复合物分别先后和同时接种于PAM细胞,分别于12、24、36、48h收集细胞液,同时设PRRSV感染对照组、免疫复合物对照组和健康细胞对照组,利用建立的相对荧光定量PCR和绝对荧光定量PCR方法分别检测巨噬细胞中IFN-α、IL-10和TNF-α的mRNA转录水平及PRRSV的RNA水平,并进行定量分析,同时采用ELISA方法检测健康细胞中IFN-α的蛋白水平。结果显示,在感染后12～36h期间,免疫复合物能够抑制PRRSV诱导的IFN-α的mRNA水平,TNF-αmRNA水平在感染后12h被促进,而在24～36h期间被抑制。在感染后48h,免疫复合物能促进IFN-α和TNF-α的mRNA水平,而在12～48h期间均能抑制IL-10的mRNA水平,此外,免疫复合物在感染12、24和36h时间段内均能明显促进病毒的增殖,但在48h后不显著。健康细胞中IFN-α的蛋白水平在培养12～48h之内呈"降-升-降"的趋势。结果表明,IFN-α蛋白的表达与其mRNA的转录不同步。在PRRSV感染初期,免疫复合物能抑制PRRSV诱导的抗病毒因子的mRNA水平,病毒的复制被促进,而在感染后期,抗病毒因子达到一定的浓度后发挥抗病毒作用,在一定程度上抑制病毒的复制。而TNF-α的转录规律不明显,表明免疫复合物可能同时与激活型和抑制型FcγR结合来共同调节PRRSV诱导的细胞因子的转录。

To research the mechanism of porcine reproductive and respiratory syndrome virus （PRRSV） infection mediated by immune complex, we investigated the effects of immune complex on the factors which affect the multiplication of PRRSV. In this study, 200 TCIDs0was inoculated in PAM cells, with an equal volume rabbit anti-pig IgG-IgG complex, which final concentration was 1.30 mg · mL^-1 , and was added to the PAM cells before, after or with PRRSV infection. Cells and the supernatant were collected at 12, 24, 36 and 48 hours, respectively. While we also set the control group with PRRSV infection, immune complexes group and the healthy cells group, apart. The mRNA levels of IFN-α IL-10 and TNF-α were detected by establishing relatire quantitative PCR method. The PRRSV RNA copies of 48 hours were detected by establishing absolute quantitative PCR method. And the quantitative data was analysed. At the same time, the protein quantity of IFN-a was detected by the ELISA method. The immune complex could en- hance the multiplication of PRRSV in PAM absolutely （P〈0.05）. At the same time, after the infection of 48 hours, the immune complex could raise the mRNA levels of IFN-a and TNF-α, but before the 36 hours, it had no evident regulation, while in all the times, the immune complex could decrease the mRNA level of IL-10. The protein level of INF-a is showed as a ＂down-up- down＂ tendency in healthy PAM cells during 12 to 48 hours. The result indicated that the expres- sion and transcription of IFN-a was not happened at the same time. While PRRSV infected at the early stage, the immune complex could suppress the transcription levels of antiviral cytokines in- duced by PRRSV infection, and the virus’ copy was promoted. But at the later, the enhancement of PRRSV was inhibited owing to the high concentration of antiviral cytokines. And the tran- scription of TNF-α induced by the immune soned for that the immune complex could jointly regulate the cytokines transcription complex had no obvious regulation. It might be rea- combine with both activated and inhibitory FcTR to induced by PRRSV.