PCV2通过激活MyD88调控体外培养PAMs分泌IL-1β、IL-6和IL-10

doi:10.11843/j.issn.0366-6964.2014.04.020

摘要/Abstract

摘要：

试验选取6头猪圆环病毒2型（PCV2）和猪繁殖与呼吸综合征病毒（PRRSV）抗原、抗体均为阴性的30日龄断奶仔猪，无菌分离肺泡巨噬细胞（AMs），分4组进行体外培养：对照组、髓样分化因子88（MyD88）干扰组（干扰组）、PCV2感染组（PCV2组）、PCV2感染-MyD88干扰组（PCV2-干扰组）。采用小干扰RNA(siRNA)方法使MyD88基因沉默，并分别于培养后0、6、12、24和48 h收集细胞及培养上清液。用荧光定量PCR法检测干扰效率，Western Blot方法检测细胞内MyD88蛋白的表达变化，ELISA方法检测培养上清液中IL-1β、IL-6和IL-10的动态变化。结果显示，siRNA 能使78%的MyD88基因沉默，并显著影响其蛋白表达；AMs中MyD88表达量，PCV2组从6 h开始显著升高（P<0.05），PCV2-干扰组与PCV2组相比在12、24、48 h差异极显著（P<0.01）。培养后各时间点，PCV2均能明显促进AMs分泌IL-1β（P<0.01或P<0.05），PCV2-干扰组和PCV2组相比，IL-1β的分泌量均显著降低（P<0.05）。PCV2能显著增加感染后6和12 h IL-6的分泌量（P<0.01），PCV2-干扰组和PCV2组相比，IL-6的分泌量在6和12 h显著降低（P<0.05）。PCV2明显促进感染后12、24和48 h AMs分泌IL-10的能力（P<0.01），PCV2-干扰组和PCV2组相比，IL-10的含量在24和48 h均极显著的降低（P<0.01）。结果表明，PCV2可引起体外培养的PAMs分泌 IL-1β、IL-6和IL-10发生不同的变化，而MyD88是引起这些变化的重要调节因子。

Abstract:

Six 30-day-old piglets (post-weaned) were selected as experiment animals，which were free of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) antibody and antigen as detected by ELISA and RT-PCR.The alveolar macrophages (AMs) isolated aseptically were divided into four groups，i.e.，control group，molecular myeloid differentiation 88 (MyD88) interference group (interference group)，PCV2 infection group (PCV2 group) and PCV2 infection-MyD88 interference group (PCV2-interference group)，and cultured in vitro.The MyD88 gene was knocked down by siRNA (small interfering RNA).After PCV2 infection，the cells and the cultural supernatants were collected at 0，6，12，24 and 48 h respectively.The gene-silencing efficiency of siRNA was detected by fluorescence quantitative PCR.The intracellular expression of MyD88 protein was detected by Western blot.The dynamic changes of IL-1β，IL-6 and IL-10 in the supernatants were assayed by ELISA.The results showed that siRNA could lead to 78% of the MyD88 gene silencing，and significantly affected the expression of MyD88 protein (P<0.01).The amount of MyD88 expressed in AMs in PCV2 group increased significantly after 6 h (P <0.05)，which was significantly higher compared to that in PCV2-interference group (P <0.01) at 12，24 and 48 h.The yield of IL-1β secreted in AMs at 6 h infected with PCV2，was notably higher compared with those in control group (P<0.01) and PCV2-interference group (P<0.05).The level of the secreted IL-6 in the PCV2 group was significantly higher than those in the control group (P<0.01) and PCV2-interference group (P<0.05) at 6 and 12 h.The yield of IL-10 secreted in AMs significantly increased (P<0.01) at 12，24 and 48 h infected with PCV2; the yield of IL-10 in PCV2-interference group significantly decreased (P<0.01) at 24 and 48 h.The results indicated that the infection of PCV2 contributed to different changes of the secretion of IL-1β，IL-6 and IL-10 in PAMs cultured in vitro，where MyD88 was involved as an important regulator.

中图分类号:

S852.33

韩俊源，郭华，张亚群，段滇宁，李晓琳，陈萌萌，张书霞. PCV2通过激活MyD88调控体外培养PAMs分泌IL-1β、IL-6和IL-10[J]. 畜牧兽医学报, 2014, 45(4): 647-653.

HAN Jun-yuan，GUO Hua，ZHANG Ya-qun，DUAN Dian-ning，LI Xiao-lin，CHEN Meng-meng，ZHANG Shu-xia. PCV2 Regulates PAMs Secreting IL-1β，IL-6 and IL-10 through the Activation of MyD88 in vitro[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(4): 647-653.

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