Laboratory of Immunology and Molecular Parasitology, Department of Microbiology and Medical Zoology, School of Medicine, University of Puerto Rico, San Juan, P.R., Puerto Rico.
Vet Parasitol. 2010 Apr 19;169(1-2):82-92. doi: 10.1016/j.vetpar.2009.12.018. Epub 2009 Dec 22.
Use of the rabbit as disease model has long been hampered by a lack of immunological assays specific to this species. In the present study we developed a SYBR Green-based, real-time RT-PCR protocol to quantitate cytokine mRNA in freshly harvested rabbit peripheral mononuclear cells. The method was validated in the course of a vaccination trial in which animals vaccinated with the recombinant antigen FhSAP2 were challenged with Fasciola hepatica metacercariae. Changes in the levels of rabbit interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha (TNFalpha), and interferon-gamma (IFNgamma) mRNA were determined. Messenger RNA from the universally expressed housekeeping gene GAPDH was used as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. Rabbits vaccinated with FhSAP2 showed an 83.3% reduction in liver fluke burden after challenge infection when compared to non-vaccinated controls. All cytokine mRNAs were found at detectable levels; however, the levels of IFNgamma, TNFalpha, IL-2 and IL-10 were significantly higher in the vaccinated group compared to the non-vaccinated group. These results suggest that protection conferred by FhSAP2 protein could be associated with a mixed Th1/Th2 immune response in which Th1 cytokines are dominant. The real-time RT-PCR method described herein can be a useful tool for monitoring changes in basic immune functions in the rabbit model of fascioliasis and may also aid in studies of human diseases for which the rabbit is an important experimental model.
利用兔子作为疾病模型一直受到缺乏针对这种物种的特异性免疫测定方法的限制。在本研究中，我们开发了一种基于 SYBR Green 的实时 RT-PCR 方案，用于定量新鲜收获的兔外周单核细胞中的细胞因子 mRNA。该方法在疫苗接种试验中得到了验证，在该试验中，用重组抗原 FhSAP2 接种的动物用 Fasciola hepatica 囊蚴进行了挑战。测定了兔白细胞介素 (IL)-2、IL-4、IL-6、IL-10、肿瘤坏死因子-α (TNFalpha) 和干扰素-γ (IFNgamma) mRNA 的水平。普遍表达的管家基因 GAPDH 的信使 RNA 用作扩增对照，并允许校正 RNA 提取和逆转录效率的变化。与未接种对照相比，用 FhSAP2 接种的兔子在挑战感染后肝片吸虫负担减少了 83.3%。所有细胞因子 mRNA 均以可检测水平存在；然而，与未接种组相比，接种组的 IFNgamma、TNFalpha、IL-2 和 IL-10 水平明显更高。这些结果表明，FhSAP2 蛋白提供的保护可能与 Th1/Th2 混合免疫反应有关，其中 Th1 细胞因子占优势。本文所述的实时 RT-PCR 方法可作为监测兔 fascioliasis 模型中基本免疫功能变化的有用工具，也可辅助研究兔作为重要实验模型的人类疾病。
