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

Abstract

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

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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Impact of Concentrations and Immune-stimulating Times of Poly I:C on Gene Expression of Porcine Peripheral Blood Mononuclear Cells

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