实时荧光定量PCR检测RAW264.7巨噬细胞中Th1/Th2型细胞因子转录水平方法的建立及初步应用  

Establishment and preliminary application of a quantitative real-time PCR method for detecting the transcriptions of Th1/Th2 cytokines in RAW264. 7 cells

作　　者：单雪芹[1,2] 韩先干[2] 张敏[2] 宋军[1,2] 刘海文[2] 田明星 潘玲[1] 丁铲[2] 周锦萍[3] 于圣青[2] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]中国农业科学院上海兽医研究所,上海200241 [3]上海市动物疫病预防控制中心,上海201103

出　　处：《细胞与分子免疫学杂志》2013年第4期430-433,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点基础研究发展计划(973)资助(2010CB530202);中央级科研院所公益性研究专项(2012JB05)

摘　　要：目的建立小鼠RAW264.7巨噬细胞的Th1和Th2型细胞因子荧光定量PCR检测方法。方法依据GenBank中小鼠的β-actin基因序列、Th1型及Th2型细胞因子的核苷酸序列设计特异性引物,以RAW264.7巨噬细胞mRNA反转录后的cDNA为模板,通过构建质粒阳性标准品,建立检测上述细胞因子转录水平的real-time PCR方法。结果用建立的荧光定量PCR方法,检测牛型布鲁氏菌S2308株感染RAW264.7巨噬细胞后,巨噬细胞Th1和Th2型细胞因子的转录水平。最低检测量为102拷贝/μL,线性关系很好,R2≥0.982,该方法具有良好的特异性和重复性。结论成功建立了检测巨噬细胞中Th1和Th2型细胞因子转录水平的荧光定量PCR方法。Objective To establish a quantitative real-time PCR method to detect the transcriptions of Th1/Th2 cytokines in RAW264.？ cells. Methods The specific primers were designed according to the sequences of TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10 and β-actin in GenBank database. Total RNA was extracted from RAW264.7 cells, and then was reverse-transcriped into cDNA, which was established as a positive standard template of the quantitative real-time PCR method. The established method was used to detect the cytokine transcriptions in RAW264. ？ cells infected with Brucella abortus $2308. Results Cytokine genes above had a good linear relationship （ R2 ≥0.982） with the detection limit of 102 copies/laL standard samples. The established quantitative real-time PCR method showed high specificity, sensitivity, and single melting peak for every cytokine. Conclusion The quantitative real-time PCR method for detecting the transcription levels of cytokine Th1 and Th2 of macrophage RAW264.7 cells was established successfully.

关 键 词：TH1 TH2型细胞因子 巨噬细胞RAW264 7 实时荧光定量PCR 布鲁氏菌 

分 类 号：R392.11[医药卫生—免疫学] R378.5[医药卫生—基础医学]