中文摘要：

【目的】聚肌胞（Poly I：C）是聚肌苷酸和聚胞苷酸的共聚物,能够模拟病毒感染后所形成的ds RNA,刺激机体产生抗病毒免疫反应和炎症反应。利用Poly I：C作为免疫刺激剂对猪外周血单核细胞（Peripheral blood mononuclear cells,PBMC）的免疫刺激试验中Poly I：C的最佳作用浓度和体外免疫刺激培养时间的研究还未见报道。本研究探讨不同的Poly I：C免疫剂量和免疫刺激时间对猪PBMC各细胞因子和受体基因表达的影响,确定Poly I：C进行免疫试验的最佳作用浓度和体外免疫刺激培养时间,为利用Poly I：C进行RNA病毒感染的免疫应答调控机理研究提供试验依据。【方法】以长白仔猪为研究对象,利用分离的PBMC和1﹕5稀释的EDTA-抗凝血,设置Poly I：C浓度梯度（0、10、20和40μg·m L-1）和不同的体外免疫刺激培养时间（4、8、12和24 h）,通过荧光定量PCR方法测定Poly I：C免疫刺激下机体激活或者诱导表达的主要细胞因子（IL6,IL8,TNFα,IL10,IRF3,IFNα和IFNγ）和模式识别受体（TLR3和TLR4）的相对表达量,分析利用Poly I：C对猪全血和PBMC进行免疫刺激试验的最佳作用浓度和时间。【结果】猪PBMC中细胞因子和受体基因的表达量受Poly I：C浓度和免疫刺激时间的影响。各基因具有特征性的表达量变化曲线,达到最高表达变化倍数的浓度和体外培养时间各不相同。对于两个干扰素基因IFNα和IFNγ,最高表达量出现在Poly I：C免疫刺激培养4 h,而后随着培养时间的延长表达量逐渐降低,而其他5个细胞因子（IL6,IL8,TNFα,IL10和IRF3）和2个模式识别受体（TLR3和TLR4）基因随着Poly I：C浓度的增加和免疫刺激时间的延长变化倍数逐渐增加,在Poly I：C浓度为20-40μg·m L-1,免疫刺激时间为12-24 h达到最高表达量。但是,对于最高表达量出现在浓度20μg·m L-1和培养时间12 h的基因,其在浓度为40μg·m L-1和培养时间24 h表达变

英文摘要：

【Objective】 Polyinosinic：polycytidylic acid（Poly I：C） is a synthetic double-stranded polyribonucleotide. As an analogue of viral double-stranded RNA, Poly I：C is widely used as immunologic stimulant to study the immune regulation and inflammatory reaction of an organism to the RNA viruses. So far, no studies were reported on determination of the optional concentration and stimulating time of porcine peripheral blood mononuclear cells（PBMC） in response to Poly I：C stimulation. In the present study, we systematically analyzed the impact of Poly I：C concentrations and stimulating times on the expression of several cytokines and pattern recognition receptors and the optional concentration and stimulating time of Poly I：C in the PBMC were determined, which would provide an experimental foundation for the future use of Poly I：C and porcine PBMC to study the immune response of pigs to RNA viruses. 【Method】 Using isolated PBMC and 1：5 EDTA-anticoagulated diluted blood of three Landrace piglets, the in vitro immune stimulating experiment was performed with a series of Poly I：C concentrations（0, 10, 20 and 40 μg·m L-1） and immune-stimulating culture times（4 h, 8 h, 12 h and 24 h）. The expression of several cytokines（IL6, IL8, TNFα, IL10, IRF3, IFNα and IFNγ） and pattern recognition receptors（TLR3 and TLR4） were determined by q PCR. According to the relative expression trends of these cytokines and receptors, it was analyzed to achieve the optimal Poly I：C concentration and immune-stimulating culture time. 【Result】 The expression of the cytokines and receptors were affected by Poly I：C concentrations as well as immune-stimulating times. Each gene had its characteristic expression trend, and varied in Poly I：C concentration and immune-stimulating time to attain the highest expression. For the two interferon genes, IFNα and IFNγ, the highest expressions were observed when the stimulating time was 4 h, and their expression decreased gradually with the stimulati