猪繁殖与呼吸综合征病毒对体外培养的猪肺泡巨噬细胞分泌细胞因子的动态影响

Effect of porcine reproductive and respiratory syndrome virus on cytokines secreted by porcine alveolar macrophage cultured in vitro

为了解猪肺泡巨噬细胞(PAMs)感染猪繁殖与呼吸综合征病毒(PRRSV)后产生主要细胞因子的变化,阐明PRRS的发病机制,选取PRRSV和猪圆环病毒2型抗原和抗体均为阴性的普通断奶仔猪,分离和培养PAMs,分为试验组和对照组,试验组PAMs用PRRSV感染,对照组PAMs加等量的细胞培养液,并于感染后第0、6、12、24和36小时收集培养上清液,用间接免疫荧光法检测PRRSV感染PAMs的情况,用猪特异的ELISA试剂盒测定TNF-α、IL-1β、IL-6、IL-10的动态变化。结果显示,感染后第6小时PAMs中就能见到PRRSV,并随着感染时间的延长,病毒量逐渐增加。PRRSV感染后第6小时,PAMs分泌TNF-α的能力就极显著升高(P<0.01),然后逐渐上升,到第36小时时依然极显著高于同时间对照组(P<0.01);试验组PAMs分泌IL-1β在PRRSV作用后第6小时就明显升高(P<0.05),以后一直持续在较高水平,到第36小时仍显著高于对照组(P<0.05);PRRSV感染后,IL-6的分泌始终处在较高水平,并显著高于同时间对照组(P<0.01);PAMs分泌IL-10的能力在PRRSV感染后很快升高,然后呈现下降趋势,第6和12小时显著高于对照组(P<0.05),24h后,试验组与对照组PAMs培养上清中IL-10含量没有显著差异(P>0.05)。结果表明,PRRSV可显著影响PAMs对细胞因子的分泌,导致免疫调节功能的紊乱。

The objective of the present study was to understand the changes of main cytokines by the porcine alveolar macrophages（PAMs） after porcine reproductive and respiratory syndrome virus（PRRSV） inoculation,which may contribute to elucidating the mechanism of pathogenesis of PRRS. PAMs were col- lected from PRRSV and porcine circovirus type 2-free weaned piglets. The cells were inoculated with or without PRRSV,and the supernatants and pAMs were collected at 0,6,12,24 and 36 hours post-inocula- tion（HPI）, respectively. PAMs were collected to detect PRRSV by immunofluorescence assay（IFA）. The dynamic changes of TNF-a, IL-lfl, IL-6 and IL-10 in the supernatants were detected with corresponding ELISA kits. PRRSV was first detected in the PAMs at 6 HPI and then the amount of virus gradually in- creased. The ability of PAMs secreting TNF-a increased significantly at 6 HPI（P-~0.01） ,and then main- tained an upward trend. At 36 HPI,the level of TNF-a was significantly higher than that of mock-infected cells（P^0.01）. The ability of PAMs to secret IL-lJ？ increased significantly at 6 HPI（P%0. 05）,continued at a high level. At 3t5 l-llq, the level oI IL-I/J was stall slgnlhcantly higher than that ot mock infected cells（P〈0.05）. The ability of PAMs to secret IL-6 was always at a higher level after PRRSV in-oculation,and significantly higher than that of mock-infected cells at the same time（P〈0.01）. The ability of PAMs to secret IL-10 increased quickly after PRRSV inoculation,then showed a downward trend. At 6 HPI and 12 HPI it were significantly higher than that of mock-infected cells（P%0.05）. At 24 HPI,it had no significant differences between inoculated cells and mock-infected cells in the culture supernatant（P） 0.05）. The results showed that PRRSV inoculation could significantly affect the ability of PAMs to secret cytokines,and lead to immune dysfunction.