小鼠细胞因子实时定量PCR检测方法的建立及应用

Quantification of cytokine gene expression in mice using real-time PCR

采用荧光SYBR GreenⅠ建立小鼠细胞因子mRNA实时定量PCR的检测方法；并利用建立的实时定量PCR的检测方法对感染H5N1禽流感病毒的BALB／c小鼠不同时间采集的肺脏组织中几种重要致炎细胞因子及趋化因子mRNA表达水平进行了检测。对H5N1禽流感病毒感染的BALB／c小鼠肺脏细胞因子的检测具有高度的特异性，检测的灵敏度为10^1～10^2拷贝数。H5N1禽流感病毒感染BALB／c小鼠后，小鼠肺脏中IL-1β、IL-6、TNFα、MIG、IP-10、RANTES、MIF和HMGB1细胞因子mRNA表达水平与对照组小鼠相比均有明显差异。建立的实时定量PCR能在基因转录水平敏感和特异地反应细胞因子的表达水平，该技术在基础和临床免疫研究中，具有良好的应用价值。

Cytokine productions play a key role in modulation of immune responses upon virus infection or immunization. Determining cytokine profiles would be the critical to understand host immune system. To develop a real-time PCR that determine the expression levels of cytokine gene in mice. Total RNA from lungs of mice infected with H5N1 influenza virus was isolated and the cytokine mRNA profiles of IL-1β, IL-6, TNFα, MIG, IP-10, RANTES, MIF, and HMGB1 in mice infected with H5N1 influenza virus were analyzed by established real-time PCR. A method of cytokine mRNA quantification was set up with specific primers and SYBR Green Ⅰ . The cytokine mRNA of IL- 1β, IL-6, TNFα, MIG, IP-10, RANTES, MIF, and H MGB1 was obviously different in comparison with the controls. The real-time PCR is a specific,accurate and sensitive method to quantify cytokine mRNA profile and has a potential application in basic and clinical study.