MyD88siRNA诱导猪调节性树突状细胞的实验研究

MyD88 Knockdown by Liposome-mediated RNA Interference to Porcine Monocyte-derived Dendritic Cell

采用siRNA干扰猪外周血单核细胞来源DC(monocyte-derived DC,MoDC)髓样分化因子88(myeloid differentiation factor 88,MyD88)基因的表达,检测干扰前后的细胞表型,免疫学功能及生物学活性,探讨猪调节性DC诱导耐受的机制。提取猪外周血单核细胞(peripheral bloodmonocyte,PBMC),经GM-CSF和IL-4诱导,体外培养得到未成熟MoDC。通过化学合成法制备针对MyD88基因siRNA 2对,在LipofectamineTM RNAiMax转染试剂介导下转染MoDC,Westernblot法及Real-time PCR检测干扰前后MyD88基因的表达水平。培养至第五天将DC分为四组:对照组、LPS组、干扰1组和干扰2组,继续培养3天。siRNA干扰后,MyD88 mRNA和蛋白质表达水平下降>80%;MyD88siRNA序列1和序列2干扰后MoDC CD80/86、SLA-Ⅱ表达降低,MLR显示刺激指数下降,IFN-γ降低,IL-4增高,与LPS组差异均具有统计学意义(P<0.05)。脂质体介导siRNA技术能干扰猪MoDC的MyD88基因,阻断TLR信号传导途径,抑制DC成熟,获得耐受性DC,表现为对同种异体T细胞的刺激能力降低,诱导免疫反应向Th2偏移。

To interfere the swine monocyte derived dendritic cell （MoDC） with small interferon RNA （siRNA） targeting myeloid differentiation factor 88 （MyD88） to induce the porcine tolerogenic DC, then study the phenotype, immunologic and biological function of DC, immature DC （iDC） was gained by inducing PBMC with IL-4 and GM- CSE Two pairs of siRNA （siRNA1 and siRNA2） were synthesized and transfected into DC by LipofectamineTM RNAiMax controlled with siNEG transfection. Western blot and Real-time PCR were employed to examine the expression of MyD88. DCs were divided into four groups at the fifth day of culture： blank control： no stimulant; LPS group： added with LPS lug/ml; interference group （one and two）： added with LPS lug/ml after transfection by siRNA MyD88 for 8 hours. All the cells were cultured for 72 hours. Flowcytometry（FCM）was used for phenotype detection such as CD80, CD86, SLA-g. BrdU method was applied for mixed lymphocyte reaction （MLR）, and the concentrations of IFN-g and 11-4 of supernatant in MLR were measured by ELISA. Porcine MoDC was gained by inducing PBMC with IL-4 and GM-CSF. Phenotype of porcine MoDC was CDl＋CD14＋CD80＋CD86＋CD172a＋SLAII^＋; the expression of CD80, CD86 and SLAII elevated after LPS stimulation. MyD88 mRNA and protein level reduced to 80% in both siRNA groups compared with siNEG group, while expression of CD80, CD86 and SLAII decreased in siRNA1 and siRNA2 groups. Stimulating index （SI） in siRNA group was reduced compared with LPS group （P〈0.05）, but no significant difference between two groups. The concentrations of IFN-g and IL-4 in supernatant of MLR of LPS group slightly increased compared with blank control （P〉0.05）. IFN-g decreased and IL-4 increased in both siRNA groups with comparison to LPS group （P〈0.05）. The siRNA transfected by liposome was able to interfere the expression of MyD88 in porcine DC by which TLR signal transduction was blocked to inhibit the maturation of DC. This tolerogenic DC was weaker stimulant for allogenic T cells and induce the deviation to Th2.