摘要
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)抗原及抗体均为阴性的普通断奶仔猪,无菌取脾脏,制成单个细胞悬液进行体外培养,并分为PCV2组和对照组,PCV2组用PCV2与脾细胞共同培养,对照组加等量RPMI-1640培养液。分别于培养0、6、12、24和48 h后收取悬浮的淋巴细胞,用间接免疫荧光法检测PCV2感染淋巴细胞的情况,相对定量RT-PCR法检测淋巴细胞中TLR2、TLR3、TLR4、TLR7、TLR8和TLR9 mRNA的表达量,Western blot检测TLR3和TLR4的蛋白表达情况。结果显示:PCV2感染后,TLR2和TLR9 mRNA的表达量在12、24和48 h时均极显著高于对照组(P<0.01),TLR3和TLR4 mRNA的表达量在24 h时极显著高于对照组(P<0.01)。TLR3蛋白在6和12 h显著高于对照组(P<0.05),TLR4蛋白在6、12、24和48 h均显著高于对照组(P<0.05)。结果表明:PCV2感染能显著上调体外培养的仔猪淋巴细胞TLR2、TLR3、TLR4和TLR9 mRNA水平,并进一步显著上调仔猪淋巴细胞TLR3和TLR4蛋白的表达,从而导致机体炎性细胞因子的分泌紊乱。
Six commercial piglets free of porcine circovirus type 2 （PCV2） and porcine reproductive and respiratory syndrome virus （PRRSV） were chosen as experiment animals. Lymphocytes isolated from spleen were randomly distributed into two groups：negative control group and PCV2-incubated group. The splenocytes of PCV2 group were incubated with PCV2 at 5×10^5 TCID50·mL^-1 ,and those of control group were incubated with equal volume of RPMI-1640. The amount of PCV2 infected cells was observed by indirect immuno- fluorescence assay （ IFA ） at 0,6,12,24 and 48 hours post infection （ hpi ）, and the mRNA expression of TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 and the protein level of TLR3 and TLR4 were detected by real-time PCR and Western blot,respectively. The results showed that the mRNA levels of TLR2 and TLR9 were higher at 12,24 and 48 h （hpi）than those of control group （P〈0. 01 ）, and those of TLR3 and TLR4 mRNA were elevated at 24 h（P〈0.01 ）. TLR3 protein contents significant increased at 6 and 12 h, and TLR4 protein contents significantly increased at 6,12,24 and 48 h（P〈0.05 ）. The results demonstrated that PCV2 significantly activate transcriptions of TLR2,TLR3,TLR4 and TLR9,and elevate the protein level of TLR3 and TLR4 in piglet lymphocytes in vitro,which mediated the produc- tion of correlative inflammatory cytokines. This may be associated with the disorder secretion of inflammatory cytokines caused by PCV2.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2013年第5期93-98,共6页
Journal of Nanjing Agricultural University
基金
国家自然科学基金项目(31172292)
