Budhia Sangeeta, Haring Lisa F, McConnell Ian, Blacklaws Barbara A
Department of Veterinary Medicine, University of Cambridge, Cambridge, CB3 0ES, United Kingdom.
J Immunol Methods. 2006 Feb 20;309(1-2):160-72. doi: 10.1016/j.jim.2005.12.006. Epub 2006 Jan 10.
In this study we describe for the first time the dynamics of the expression of the cytokines, IL-1beta, IL-12p40, TNFalpha in ovine dendritic cells and macrophages after LPS stimulation. Real time RT-PCR was used for the quantitation of these cytokines and IL-4 and IFNgamma as well as two potential housekeeping genes (HKG), ATPase and GAPDH, in mRNAs from ovine leucocyte populations. Both dual-labelled probes (TAMRA/FAM) and SYBR Green assays were utilised, using a Corbett Research RotorGene and ABI 7700 machine. In order to quantitate each cytokine in our assays all C(T) values were compared to a standard curve generated using plasmid DNA containing the cytokine of interest. To validate our assays, concanavalin A-stimulated peripheral blood mononuclear cells (PBMCs) and LPS-stimulated monocyte-derived dendritic cells (MoDC) and monocyte-derived macrophages (MDMØ) were examined. We found that peak cytokine mRNA expression was between 3 and 6 h for the cytokines examined except for IL-12p40 where peak cytokine release was around 12 h post-stimulation in MDMØ and PBMCs. However, in MoDCs, peak IL-12p40 mRNA expression was observed within 3-6 h. We have identified a sensitive and reliable method for the identification of ovine cytokine mRNAs.
在本研究中，我们首次描述了脂多糖刺激后绵羊树突状细胞和巨噬细胞中细胞因子白细胞介素-1β（IL-1β）、白细胞介素-12 p40（IL-12p40）、肿瘤坏死因子α（TNFα）表达的动态变化。采用实时逆转录聚合酶链反应（RT-PCR）对绵羊白细胞群体mRNA中的这些细胞因子以及白细胞介素-4（IL-4）、干扰素γ（IFNγ）和两个潜在的看家基因（HKG）——ATP酶和甘油醛-3-磷酸脱氢酶（GAPDH）进行定量分析。使用Corbett Research RotorGene和ABI 7700仪器，同时采用双标记探针（TAMRA/FAM）和SYBR Green检测法。为了在我们的检测中对每种细胞因子进行定量，将所有的C(T)值与使用含有目标细胞因子的质粒DNA生成的标准曲线进行比较。为验证我们的检测方法，对伴刀豆球蛋白A刺激的外周血单核细胞（PBMC）以及脂多糖刺激的单核细胞衍生树突状细胞（MoDC）和单核细胞衍生巨噬细胞（MDMØ）进行了检测。我们发现，除IL-12p40外，所检测细胞因子的mRNA表达峰值在3至6小时之间，在MDMØ和PBMC中，IL-12p40的细胞因子释放峰值在刺激后约12小时左右。然而，在MoDC中，IL-12p40 mRNA表达峰值在3 - 6小时内出现。我们已经确定了一种灵敏且可靠的方法来鉴定绵羊细胞因子mRNA。
