3种不同配体及其组合对鸭树突状细胞TLRs信号通路和细胞因子表达的影响

Effects of Three Different Ligands Alone or Combined on the TLRs Signaling Pathway and Cytokines Expression in Duck Dendritic Cells

为研究不同Toll样受体(Toll like receptor,TLR)配体及其组合对鸭外周血单核源树突状细胞(Peripheral blood monocyte-derived dendritic cells,MoDCs)TLRs信号通路和细胞因子表达的影响,该研究体外分离、诱导获得鸭MoDCs,对其形态学特征和膜表面标记分子进行观察、鉴定,并将TLR2配体Pam2CSK4、TLR3配体Poly(I∶C)和TLR7配体Imiquimod单独或组合刺激鸭MoDCs 12 h和24 h,通过荧光定量PCR方法检测鸭MoDCs中TLRs信号通路关键节点蛋白和免疫相关因子的表达情况。结果显示,培养至第7-8天,所获得的鸭MoDCs呈现典型树突状细胞形态。刺激24 h后,核转录因子κB(NF-κB)在Imiquimod、Pam2CSK4+Imiquimod和Poly(I∶C)+Imiquimod组高表达,但这三组之间差异不显著。与对照组和TLR单独配体组相比,IL-2和IL-6 m RNA在Pam2CSK4+Imiquimod和Poly(I∶C)+Imiquimod组高表达,IFN-γ在Pam2CSK4+Imiquimod组高表达,而IFN-α和MHC分子则在Poly(I∶C)+Imiquimod组高表达。结果表明Pam2CSK4、Poly(I∶C)和Imiquimod单独或不同组合均可有效激活鸭MoDCs TRIF/MyD88-NF-κB信号通路,提示Pam2CSK4+Imiquimod和Poly(I∶C)+Imiquimod组合都具有一定的免疫激活协同效应,但Poly(I∶C)+Imiquimod组合可以更好地促进鸭MoDCs的成熟。

In order to study the effects of different Toll⁃like receptor(Toll like receptor,TLR)ligand alone or in combinations on TLRs signaling pathway and cytokines in duck peripheral blood monocyte⁃derived dendritic cells(MoDCs).In this study,duck MoDCs were isolated and cultured,and the morphological characteristics and membrane surface marker molecules were observed and identified.The gene mRNA expression of key factors related to TLRs signaling pathway in duck MoDCs were detected by fluorescent quantitative PCR after the duck MoDCs were stimulated with TLR2 ligand Pam2CSK4,TLR3 ligand Poly(I∶C)and TLR7 ligand Imiquimod alone or in combination for 12 h and 24 h,respectively.The results showed that the duck MoDCs showed typical dendritic cell morphology on the 7th to 8th day of culture.After 24 h’s incubation,NF⁃κB was highly expressed in Imiquimod,Pam2CSK4+Imiquimod and Poly(I∶C)+Imiquimod groups,but there was no significant difference between the three groups.Compared with the control group and TLR alone ligand group,the highly expressed IL⁃2 and IL⁃6 were obsereved in Pam2CSK4+Imiquimod and Poly(I∶C)+Imiquimod groups,and IFN⁃γshowed in Pam2CSK4+Imiquimod group,while IFN⁃αand MHC molecules were observed in Poly(I∶C)+Imiquimod group.The results showed that Pam2CSK4,Poly(I∶C)and Imiquimod alone or in different combinations could effectively activate TRIF/MyD88⁃NF⁃κB signal pathway in duck MoDCs.The results of the presetn study suggested that both of Pam2CSK4+Imiquimod and Poly(I∶C)+Imiquimod combination have a certain synergistic effect of immune activation,but Poly(I∶C)+Imiquimod combination is able to promote the maturation of duck MoDCs.