PRRSV体外感染Marc-145细胞对NF-κB p65/p50转录时相及核易位的影响

Influence of PRRSV infection to Marc-145 cells in vitro on NF-κB p65/p50 transcription phase and nuclear translocation

为了解猪繁殖与呼吸综合征病毒(PRRSV)感染对细胞核转录因子-κB(NF-κB,p65/p50)mRNA转录时相及核易位的影响,探讨PRRSV感染导致的免疫抑制与NF-κB的活化异常是否有一定的相关性,运用实时荧光定量PCR技术对PRRSV感染Marc-145细胞不同时期NF-κB p50、p65mRNA的转录水平变化进行定量分析,并提取不同感染时期Marc-145细胞核蛋白,利用免疫印迹法检测细胞核内具有活性的p50、p65水平变化。荧光定量结果表明,Marc-145细胞感染PRRSV后第8～12小时,p50、p65转录水平急剧下调,第12～120小时p50、p65mRNA含量一直维持在较低水平,与接毒前及对照组同时期的转录水平相比,差异显著或极显著(P<0.05或P<0.01)。免疫印迹结果表明,Marc-145细胞感染PRRSV后第12～96小时,细胞核内均未检测到p50的表达;感染后第12小时,细胞核内p65表达下调,第48～96小时未检测到p65的表达。可见,PRRSV体外感染Marc-145细胞能够抑制NF-κB p65/p50mRNA的转录及核易位。以上结果支持PRRSV感染早期无力的先天性免疫应答可能与NF-κB活化水平的低下有关的观点,为进一步研究PRRS的感染机制及NF-κB的功能提供新的思路和依据。

To study the influence of PRRSV infection on mRNA transcription phase and nuclear translocation of nuclear factor-κB（NF-κB,p65/p50） in vitro,and to investigate whether PRRSV induces immunologic suppression related to NF-κB abnormal activation,the p65/p50 genes mRNA transcription levels of Marc-145 cells infected with PRRSV in different periods were analyzed using real-time fluorescence quantitative PCR（real-time FQ-PCR）.Nuclear proteins of Marc-145 cells infected with PRRSV in different periods were extracted,and the activity levels of p65/p50 in the cellular nuclear were detected by Western-blot.Real-time FQ-PCR results indicated that,the p65/p50 mRNA transcription levels of Marc-145 cells decreased rapidly at 8-12 hour post-infection（hpi）,then kept at a low level at 12-120 hpi,which was significantly lower than the levels of control group at the same time and Marc-145 cells without PRRSV infection（P〈0.05 or P〈0.01）.The p50 expression in the celluar nuclear could not be detected at 12-96 hpi by Western-blot,neither the p65 at 12 hpi.The results revealed that PRRSV infection in vitro would inhibit NF-κB p65/p50 mRNA transcription and nuclear translocation.The PRRSV-induced immunologic suppression may be related to the low activation level of NF-κB.This study would provide some new ideas for further studies on the pathogenic mechanism of PRRSV and the function of NF-κB.