摘要
检测猪圆环病毒2型(PCV2)对体外培养仔猪淋巴细胞核因子-κB(NF-κB)信号的动态影响,探讨PCV2调控仔猪淋巴细胞炎性细胞因子产生与变化的机制。选取5头PCV2和猪繁殖与呼吸综合征病毒(PRRSV)血清抗体和抗原阴性的普通断奶仔猪,无菌采集脾脏,分离脾脏细胞制成单个细胞悬液进行体外培养。分为对照组和试验组,试验组每毫升细胞悬液加PCV2病毒(病毒滴度为5×105 TCID50.mL-1)悬液200μL;对照组加等量1640细胞培养液,并分别于培养后0、6、12、24h收取悬浮的淋巴细胞。激光共聚焦显微镜检测NF-κB核易位,WesternBlot检测核蛋白中NF-κB/P65、胞质蛋白中磷酸化抑制性κBα(p-IκBα)含量,电泳迁移率(EMSA)法检测NF-κB与DNA的结合率。结果如下:淋巴细胞中NF-κB/P65蛋白核易位随着PCV2作用时间的延长而增加;淋巴细胞核中NF-κB/P65蛋白随PCV2作用时间增加逐渐升高,6h后均显著高于同时间对照组(P<0.05);NF-κB与DNA的结合率也随PCV2作用时间增加逐渐升高,PCV2作用6h后均显著高于对照组(P<0.05);淋巴细胞胞质中的p-IκBα蛋白水平在PCV2作用24h时达到峰值,并且培养6、12和24h均极显著高于同时间对照组(P<0.01)。研究表明PCV2可通过IκB的磷酸化降解途径显著激活仔猪淋巴细胞中NF-κB信号,使其发生核易位并与DNA发生结合,从而调控相关炎性细胞因子的表达。
The effects of PCV2 on NF-κB signal were studied to explore the modulation mechanism of PCV2 on release and changes of pro-inflammatory factors from lymphocytes in PCV2 infected piglets. Five conventional post-weaning piglets free of PCV2 and PRRSV antibody and antigen were chosen as the experiment animals. The cells isolated from spleens with pinhead of injectors were distributed into experimental group and control group. The lymphocytes of experimental group were incubated with PCV2 at 1 × 105 TCID50, control group was incubated with equal volume of 1640. The PCV2-inoculated and mock-inoculated lymphocytes at 0, 6, 12, and 24 h post-inoculation （HPI） were collected respectively. NF-κB was translocated into nuclear by confocal laser scanning microscope. The protein levels of NF-κB/P65 and phosphorylation-IκBα were detected by Western Blot and NF-κB DNA binding activity was detected by electrophoretic mobility shift assays （EMSA）. The result of confocal laser scanning microscope showed that nuclear translocation of NF-κB/P65 after lymphocytes incubation with PCV2 increased gradually with the increased incubation time. The NF-κB/P65 proteins increased significantly （P〈0.05） in PCV2-inoculated lymphocytes compared with controls at the same time. The results of NF-κB DNA binding activity revealed the same changes with NF-κB/P65 protein. The protein levels of phosphorylation-IκBα in plasma was highest at 24HPI, and the levels in experimental group were significant increasing （P〈0.05） compared with control group at 6 HPI, 12HPI and 24 HPI. All the results demonstrated that PCV2 can activate the NF-κB signaling pathway through the degradation of IκB phosphorylation, and the signal could play an important role in modulating the expression of the some pro-inflammation cytokines.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2013年第5期760-766,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31172292
30871848)
