聚肌胞浓度和免疫刺激时间对猪外周血单核细胞基因表达的影响

doi:10.3864/j.issn.0578-1752.2015.03.17

摘要/Abstract

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dsRNA，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40 μg·mL^-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20-40 μg·mL^-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20 μg·mL^-1和培养时间12 h的基因，其在浓度为40 μg·mL^-1和培养时间24 h表达变化量只有小幅下降。另外本研究还对比检测了1﹕5稀释血在Poly I:C免疫刺激下主要细胞因子和受体的表达变化，结果表明全血中各细胞因子和受体的整体表达变化倍数比PBMC低很多，特别是对于IL6和IL8这两个PBMC中表达变化很大的基因，全血中表达变化量不仅很小，而且变化趋势相反。相对于PBMC，稀释全血完整地保存了机体的原始状态，但是利用全血进行免疫试验的主要缺点是抗凝剂保留在全血中。研究中EDTAK2抗凝剂能与血液中的钙离子结合成为螯合物，从而阻止血液凝固，但是钙离子是细胞的重要信号分子，钙离子与EDTA的结合对后续的细胞功能可能产生一定的影响。另外，全血中的某些化合物与血浆蛋白的等成分可能参与免疫细胞对Poly I:C免疫刺激的调节，减少了对Poly I:C免疫刺激的应答。【结论】（1）综合分析所检测的细胞因子和受体的变化趋势，利用Poly I:C进行猪PBMC免疫刺激试验的最佳浓度和体外培养时间分别为20 μg·mL^-1和24 h。（2）1﹕5稀释的EDTA-抗凝血对Poly I:C免疫反应反应小，与PBMC对Poly I:C免疫应答有不同的应答反应趋势。

关键词: 猪, 聚肌胞（Poly I:C）, 外周血单核细胞（PBMC）, 基因表达

Abstract: 【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·mL^-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 qPCR. 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 stimulating time. For the other five cytokines (IL6, IL8, TNFα, IL10 and IRF3) and two receptor genes (TLR3 and TLR4), the expressions were gradually increased with the Poly I:C concentrations and stimulation times, and reached the highest when Poly I:C concentrations and stimulation times were 20-40 μg·mL^-1 and 12-24 h, respectively. However, for the genes whose expressions were highest when Poly I:C concentration and stimulation time were 20 μg·mL^-1 and 12 h, their expressions showed small decline when Poly I:C concentration and stimulation time were 40 μg·mL^-1 and 24 h. Furthermore, in the study, it was also tested the expressions of the above genes of 1:5 EDTA-anticoagulated diluted blood in response to various Poly I:C concentrations and stimulation times. The results indicated that the whole expressions of these genes were lower than those in PBMC, especially for two interleukin genes, IL6 and IL8, whose expression in diluted whole blood was not only low but the variation trends with Poly I:C concentrations and stimulation time were reversed with those in PBMC. Compared with PBMC, though diluted blood preserved the integrity of the immune state of the pig’s blood, the major drawback is that the anticoagulant is kept in it. The anticoagulant used in the study was EDTAK2, which can combine with calcium ion to form chelate, so as to prevent blood clotting. However, calcium ion is an important signal molecule in cells, and the combination of calcium ion with EDTA may have a certain impact on the subsequent cell function. In addition, some of the compounds and plasma proteins in whole blood may be also involved in the regulation of immune cells to Poly I:C immune stimulation so as to reduce immune response. 【Conclusion】 Comprehensive analysis of the expression trends of the cytokines and receptors were detected, and it was concluded that, in the porcine PBMC stimulation experiment using Poly I:C, the optimal concentration and immune-stimulating time are 20 μg·mL^-1 and 24 hours, respectively. EDTA- anticoagulated diluted blood has less immune response to Poly I:C and different variation trends with Poly I:C concentrations and stimulation times.

Key words: pig, polyinosinic-cytidylic acid (Poly I:C), porcine peripheral blood mononuclear cell (PBMC), gene expression

王继英，王彦平，王怀中，王海飞，刘剑锋，郭建凤. 聚肌胞浓度和免疫刺激时间对猪外周血单核细胞基因表达的影响[J]. 中国农业科学, 2015, 48(3): 583-593.

WANG Ji-ying, WANG Yan-ping, WANG Huai-zhong, WANG Hai-fei, LIU Jian-feng, GUO Jian-feng. Impact of Concentrations and Immune-stimulating Times of Poly I:C on Gene Expression of Porcine Peripheral Blood Mononuclear Cells[J]. Scientia Agricultura Sinica, 2015, 48(3): 583-593.

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