小反刍兽疫病毒Nigeria75/1疫苗毒及其N蛋白对山羊PBMCs免疫效应的影响

Effects of Peste Des Petits Ruminants Virus Nigeria 75/1 Vaccine Strain and NProtein on the Immune Responses of Goat Peripheral Blood Mononuclear Cells

小反刍兽疫(PPR)是羊、骆驼等小反刍动物的一种急性、烈性、接触性A类传染病,发病率和致死率极高。目前,PPR在全球仍呈现区域性流行和多地散发势态。为探讨PPRV及N蛋白体外诱导山羊外周血单个核细胞(PBMCs)在不同时间对PBMCs免疫应答效应的影响。本研究将PPRV Nigeria75/1疫苗毒(1 MOI)、重组N蛋白(10μg/mL)和RPMI 1640(阴性对照)体外刺激PBMCs 48h、72h、96 h。采用CCK⁃8法检测PBMCs细胞增殖情况;qRT⁃PCR及ELISA检测炎症因子包括IL⁃1β、IL⁃6、IL⁃10、TNF⁃α和IFN⁃γ的mRNA表达水平及分泌情况;流式细胞术检测T细胞CD4+和CD8+的表达、及单核来源树突状细胞(DCs)表面分子CD40、CD86、CD80的表达、以及检测PPRV感染PBMCs引起的细胞凋亡。研究发现:与对照组相比,PPRV能够抑制PBMCs的体外增殖,显著促进炎症因子IL⁃1β、IL⁃6、IL⁃10、TNF⁃α、IFN⁃γ的表达(P<0.05)。并且PPRV感染PBMCs产生细胞凋亡,促进CD4+T细胞和CD8+T细胞表达。另外PPRV体外刺激DCs,CD40、CD86、CD80的表达显著升高(P<0.05),提示PPRV具有刺激DCs细胞成熟与分化的功能。进一步研究发现PPRV N蛋白体外刺激PBMCs能引起与PPRV作用相同的免疫效应。本研究表明PPRV Nigeria75/1疫苗毒体外感染PBMCs主要引起炎症反应与细胞凋亡、促进单核来源DCs成熟与分化,并且N蛋白参与PPRV引起的各项免疫功能。

Peste des petits ruminants(PPR)is an acute,severe and contagious disease that mainly infects sheep,camels and other small ruminants,with a high morbidity and mortality.At present,PPR still appears with a regional epidemic and regional distribution in the world.In order to investigate the immune response effects of goat peripheral blood mononuclear cells(PBMCs)after infection with PPRV and stimulation with N protein at different time in vitro.We used PPRV Nigeria75/1 vaccine strain(MOI=1),recombinant N protein(10μg/mL)and RPMI1640 media(negative control)to stimulate PBMCs for 48,72,96 h.CCK⁃8 method was used to detect cell proliferation.The expression levels of IL⁃1β,IL⁃6,IL⁃12,the TNF⁃αand IFN⁃γwere determined by qRT⁃PCR and ELISA.Besides,flow cytometry was used to investigate the expression of CD4+and CD8+T cells,the expression of CD40,CD86,and CD80 from monocyte⁃derived dendritic cells(DCs),as well as apoptosis of PBMCs.The results showed that compared with mock cells,when goat PBMCs were infected with PPRV,the mRNA expression levels and secretion of IL⁃6,IL⁃1β,IL⁃12,IFN⁃γand TNF⁃αwere increased.Moreover,PPRV stimulated the differentiation of CD4+and CD8+T cells,and increased the expression of CD40,CD80 and CD86 in monocytederived dendritic cells(DCs),suggesting that PPRV could stimulate the maturation and differentiation of DCs.Meanwhile,PPRV induced apoptosis and inhibited cell proliferation.Furthermore,we found that stimulation of PBMCs in vitro with PPRV N protein can induce the same immune effect as that of PPRV.This study demonstrated that PPRV Nigeria75/1 vaccine strain infection with PBMCs mainly caused inflammation and apoptosis,promoted the maturation and differentiation of DCs,and N protein was involved in various immune functions induced by PPRV.