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

PCV2 Regulates PAMs Secreting IL-1β,IL-6 and IL-10 through the Activation of MyD88 in vitro

作　　者：韩俊源[1] 郭华[1] 张亚群[1] 段滇宁[1] 李晓琳[1] 陈萌萌[1] 张书霞[1] 

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《畜牧兽医学报》2014年第4期647-653,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31172292)

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48h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6h开始显著升高(P<0.05),PCV2-干扰组与PCV2组相比在12、24、48h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12hIL-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12h显著降低(P<0.05)。PCV2明显促进感染后12、24和48hAMs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48h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。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),PCV2infection group(PCV2 group)and PCV2infection-MyD88interference group(PCV2-interference group),and cultured in vitro.The MyD88gene was knocked down by siRNA(small interfering RNA).After PCV2infection,the cells and the cultural supernatants were collected at 0,6,12,24and 48hrespectively. The gene-silencing efficiency of siRNA was detected by fluorescence quantitative PCR.The intra-cellular expression of MyD88protein was detected by Western blot.The dynamic changes of IL-1β,IL-6and IL-10in the supernatants were assayed by ELISA.The results showed that siRNA could lead to 78% of the MyD88gene silencing,and significantly affected the expression of MyD88protein(P<0.01).The amount of MyD88expressed in AMs in PCV2group increased significantly after 6h(P <0.05),which was significantly higher compared to that in PCV2-interference group(P <0.01)at 12,24and 48h.The yield of IL-1βsecreted in AMs at 6hinfected 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-6in the PCV2group was significantly higher than those in the control group(P<0.01)and PCV2-interference group(P<0.05)at 6 and 12h.The yield of IL-10secreted in AMs significantly increased(P<0.01)at 12,24and 48h infected with PCV2;the yield of IL-10in PCV2-interference group significantly decreased(P< 0.01)at 24and 48h.The results indicated that the infection of PCV2contributed to different changes of the secretion of IL-1β,IL-6and IL-10in PAMs cultured in vitro,where MyD88was involved as an important regulator.

关 键 词：猪圆环病毒2型 猪肺泡巨噬细胞 髓样分化因子88 细胞因子 

分 类 号：S858.28[农业科学—临床兽医学]