猪圆环病毒2型感染仔猪肺泡巨噬细胞Toll样受体mRNA转录的变化

Changes of Toll-like Receptor mRNA Transcription in Alveolar Macrophages of Piglets Infected with Porcine Circovirus Type 2

选用6头经ELISA和荧光定量PCR方法检测猪圆环病毒2型(PCV2)和猪繁殖与呼吸综合征病毒(PRRSV)血清抗体和抗原均为阴性的普通断奶仔猪,无菌分离肺泡巨噬细胞(AMs),分为对照组和PCV2感染组(PCV2组)进行体外培养,分别在培养0、6、12、24和48h收集AMs。间接免疫荧光法定位检测PCV2感染AMs情况;荧光定量PCR方法检测TLR2、TLR3、TLR4、TLR7、TLR8和TLR9 mRNA转录量的变化。结果表明,TLR2mRNA的转录量在感染后6h迅速升高,12h后又迅速下降,但仍显著高于对照组(P<0.01或P<0.05),到48h时恢复;TLR3mRNA的转录量,攻毒后6和12h显著高于对照组(P<0.05),24和48h时恢复到对照组水平;PCV2明显升高感染后12hAMs中TLR4mRNA的转录(P<0.05),而明显抑制感染后24和48h的转录(P<0.05);PCV2引起感染后6和12h的AMs中TLR7、TLR8和TLR9mRNA转录量显著升高(P<0.01或P<0.05),而到感染后24和48h又恢复到对照组水平。结果表明,PCV2可显著影响体外培养仔猪AMs TLR2、TLR3、TLR4、TLR7、TLR8和TLR9mRNA的转录。

Six conventional piglets free of porcine circovirus type 2(PCV2)and porcine reproductive and respiratory syndrome virus(PRRSV)were used in this experiment as detected by ELISA and fluorescence quantitative PCR.The alveolar macrophages(AMs)were isolated aseptically and divided into two groups,control group and PCV2infection group(PCV2group)and cultured in vitro.The AMs were collected at 0,6,12,24and 48hours post infection(hpi).The amount of PCV2infected cells was observed by indirect immunofluorescence assay(IFA)and the mRNA transcription of TLR2,TLR3,TLR4,TLR7,TLR8and TLR9were detected by real-time PCR. The results show that the mRNA levels of TLR2were significantly higher at 6,12and 24h(hpi)(P<0.01or P<0.05),then the levels were recovered at 48hpi.The expression of TLR3were significantly elevated at 6and 12hpi(P<0.05),and the transcription were then restored at 24 and 48hpi;PCV2stimulation up-regulated the transcriptions of TLR4in AMs at 12hpi(P< 0.05)and down-regulated the transcriptions at 24and 48hpi(P<0.05);PCV2-infection increased TLR7,TLR8and TLR9transcriptions in AMs at 6and 12hpi(P<0.01or P<0.05)andtheir of transcriptions were restored at 24and 48hpi.Thus,PCV2can significantly affect TLR2, TLR3,TLR4,TLR7,TLR8and TLR9transcriptions in piglets AMs in vitro.