Listvanova Slavka, Temmerman Stéphane, Stordeur Patrick, Verscheure Virginie, Place Sammy, Zhou Lin, Locht Camille, Mascart Françoise
Laboratory of Immunology, Erasme Hospital, Université Libre de Bruxelles, Route de Lennik, 808, B-1070 Brussels, Belgium.
J Immunol Methods. 2003 Oct 1;281(1-2):27-35. doi: 10.1016/s0022-1759(03)00267-9.
Real-time polymerase chain reaction (PCR) has recently been described as a new tool to measure and accurately quantify mRNA levels. In this study, we have applied this technique to evaluate cytokine mRNA synthesis induced by antigenic stimulation with purified protein derivative (PPD) or heparin-binding haemagglutinin (HBHA) in human peripheral blood mononuclear cells (PBMC) from Mycobacterium tuberculosis-infected individuals. Whereas PPD and HBHA optimally induced IL-2 mRNA after respectively 8 and 16 to 24 h of in vitro stimulation, longer in vitro stimulation times were necessary for optimal induction of interferon-gamma (IFN-gamma) mRNA, respectively 16 to 24 h for PPD and 24 to 96 h for HBHA. IL-13 mRNA was optimally induced by in vitro stimulation after 16-48 h for PPD and after 48 to 96 h for HBHA. Comparison of antigen-induced Th1 and Th2 cytokines appears, therefore, valuable only if both cytokine types are analysed at their optimal time point of production, which, for a given cytokine, may differ for each antigen tested. Results obtained by real-time PCR for IFN-gamma and IL-13 mRNA correlated well with those obtained by measuring the cytokine concentrations in cell culture supernatants, provided they were high enough to be detected. We conclude that real-time PCR can be successfully applied to the quantification of antigen-induced cytokine mRNA and to the evaluation of the Th1/Th2 balance, only if the kinetics of cytokine mRNA appearance are taken into account and evaluated for each cytokine measured and each antigen analysed.
实时聚合酶链反应（PCR）最近被描述为一种测量和准确量化mRNA水平的新工具。在本研究中，我们应用该技术评估了结核分枝杆菌感染个体外周血单个核细胞（PBMC）中由纯化蛋白衍生物（PPD）或肝素结合血凝素（HBHA）抗原刺激诱导的细胞因子mRNA合成。PPD和HBHA分别在体外刺激8小时和16至24小时后最佳诱导IL-2 mRNA，而对于干扰素-γ（IFN-γ）mRNA的最佳诱导则需要更长的体外刺激时间，PPD分别为16至24小时，HBHA为24至96小时。PPD在体外刺激16 - 48小时后以及HBHA在体外刺激48至96小时后最佳诱导IL-13 mRNA。因此，只有在两种细胞因子类型都在其最佳产生时间点进行分析时，抗原诱导的Th1和Th2细胞因子的比较才显得有价值，对于给定的细胞因子，每种测试抗原的最佳产生时间点可能不同。实时PCR获得的IFN-γ和IL-13 mRNA结果与通过测量细胞培养上清液中细胞因子浓度获得的结果相关性良好，前提是它们足够高以被检测到。我们得出结论，只有考虑到细胞因子mRNA出现的动力学并针对每种测量的细胞因子和每种分析的抗原进行评估，实时PCR才能成功应用于抗原诱导的细胞因子mRNA的定量以及Th1/Th2平衡的评估。
