Heparin and EDTA as anticoagulant differentially affect cytokine mRNA level of cultured porcine blood cells
Heparin and EDTA as anticoagulant differentially affect cytokine mRNA level of cultured porcine blood cells
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DOI：
10.1016/j.jim.2007.04.012
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发表时间：
2007-07-31
影响因子：
2.2
通讯作者：
Gemeiner, M.
中科院分区：
文献类型：
--
作者：
Duvigneau, J. C.;Sipos, W.;Gemeiner, M.
细胞因子mRNA表达谱用于表征不同测试系统中的免疫细胞活化。稀释的全血和分离的PBMC都广泛应用于这些研究。关于不同抗凝剂用于分析细胞因子表达的适用性的全面数据对于猪不可用。因此，本研究的目的是比较两种常用的抗凝剂（肝素和EDTA）对猪血细胞细胞因子表达模式的影响。通过实时PCR在稀释的猪全血和分离的PBMC中离体和体外刺激后检测IL-1 α、IL-2、IL-4、IL-6、IL-10和IFN-γ mRNA水平。用ConA或LPS刺激细胞，已知ConA或LPS作用于不同的靶细胞并暗示不同的信号传导途径。此外，研究了分离的RNA的完整性。新鲜全血的离体细胞因子表达模式不受所研究的抗凝剂的影响。相比之下，刺激培养的稀释全血或PBMC导致显著差异，这取决于所应用的抗凝剂。使用EDTA，我们发现全血表达细胞因子的能力显著降低。然而，从EDTA抗凝血中分离的PBMC显示出比从肝素化血液中分离的PBMC更高的细胞因子表达能力。比较稀释的全血和PBMC，我们发现培养的猪全血比分离的PBMC对细菌产物的反应更好，这可能是因为存在足够的辅助血浆衍生因子，如LPS结合蛋白。然而，分离的PBMC显示出比稀释的全血更高的T细胞应答。总之，我们的研究结果强调，每个应用程序需要一个特定的检测系统。(C)2007 Elsevier B. V.保留所有权利。
Cytokine mRNA expression profiles serve to characterize immune cell activation in different test systems. Both, diluted whole blood and isolated PBMC are widely applied for these studies. Comprehensive data regarding the suitability of different anticoagulants for profiling cytokine expression are not available for the pig. Therefore the aim of this study was to compare the effect of two commonly used anticoagulants (heparin and EDTA) on the cytokine expression pattern of porcine blood cells. IL-1 alpha, IL-2, IL-4, IL-6, IL-10 and IFN-gamma mRNA levels were detected ex-vivo and upon in-vitro stimulation in diluted porcine whole blood and isolated PBMC by real-time PCR. The cells were stimulated with ConA or LPS, known to act on different target cells and implying different signalling pathways. Additionally the integrity of the isolated RNA was investigated. Ex-vivo cytokine expression pattern of fresh whole blood were not affected by the investigated anticoagulants. In contrast, stimulation of cultured diluted whole blood or PBMC resulted in significant differences depending on the applied anticoagulant. Using EDTA we found a significantly decreased capacity of whole blood to express cytokines. However, isolated PBMC from EDTA anticoagulated blood showed a higher cytokine expression capacity than PBMC from heparinized blood. Comparing diluted whole blood and PBMC we found that cultured porcine whole blood responded better to bacterial products than isolated PBMC, probably because sufficient auxiliary plasma derived factors such as LPS-binding protein, are present. However, isolated PBMC showed a higher T-cell response than diluted whole blood. In conclusion, our findings underline that each application demands a specific assay system. (C) 2007 Elsevier B.V. All rights reserved.