竞争PCR定量检测免疫小鼠细胞因子表达水平

Quantitative Measurement of Cytokine Products using Competitive PCR in Immunized Mice

目的 :利用竞争PCR技术定量检测各种细胞因子的表达水平。方法 :在机体免疫应答过程中 ,细胞因子作为免疫调节分子在免疫网络系统中发挥重要的调节功能 ,T细胞能够通过产生并分泌各种细胞因子来调节机体的免疫反应。本研究采用竞争PCR技术 ,通过提取的小鼠脾脏组织总RNA ,经反转录得到cDNA ,利用一种修饰的竞争模板pQRS进行竞争PCR扩增 ,然后利用琼脂糖凝胶电泳分析并与竞争模板产物比较而定量。结论 :结果表明竞争PCR能够初步定量免疫前后小鼠脾脏组织表达的IL- 2、IL - 4、IL - 10、IL - 12和IFN -γ的表达量。为深入研究免疫前后小鼠体内各种细胞因子的动态水平和分析细胞免疫水平奠定了基础。

Objective:To determine the expression level of native cDNA of cytokines by competitive quantification PCR.Methods:Cytokine productions play a key role in modulation of immune responses upon infection or immunization.Determining cytokine profile would be the critical to understand host immune system.To quantitatively measure the each cytokine levels from the spleens obtained before or after FMDV DNA immunized mice,the total RNA were extracted and generated into cDNA by RT reaction.The native cDNA was subjected into a competitive PCR reaction with a competitor that is a slightly larger in size.The level of native cDNA of cytokine is determined by comparison with the known amount of competitor in agarose electrophoresis.Conclusions:The results showed the competitive quantification PCR could quantify the level of IL-2,IL-4,IL-10,IL-12 and IFN-γ from the spleen of mice before and after immunization.The results from the expressions of cytokine from normal and immunized mice may be used to analyze the immune response profile or guide a better vaccine design.