摘要
目的建立细胞因子mRNA实时定量PCR的检测方法,并探讨人单个核细胞(PBMC)在激发型CD3单克隆抗体作用下,细胞因子mRNA的表达。方法分离健康个体的PBMC,体外与激发型CD3单克隆抗体共培养,应用实时定量PCR检测不同共培养时间点Th1型(IL-2,IFN-γ)和Th2型(IL-10)细胞因子mRNA的表达。结果激发型CD3抗体能显著上调PBMC胞浆IL-2,IFN-γ和IL-10mRNA的表达。在共培养的12h,IL-2mRNA的含量显著上升并达到峰值14000copies/ml,随着培养时间的延长快速下降。IFN-γ和IL-10mRNA在共培养的24～48h达到峰值,分别为110000copies/ml和15000copies/ml。不同细胞因子其胞浆mRNA水平的基础值有明显差异。结论实时定量PCR能在基因转录水平敏感和特异地反映微量生物活性因子的表达和调节。因此,该技术在基础和临床免疫研究中,具有良好的应用价值。
Objective To develop a protocol that provides an easy and accurate way to perform rapidly real-time PCR on a lightercycler instrument. This methodology was applied to the quantification of cytokine mRNA in human peripheral blood mononuelear cell（PBMC） induced by anti-D3 mAb. Methods Purified human PBMC and functional anti-CD154 mAb were co-cultured ex vivo. Total cytoplastic mRNA of PBMC stimulated by anti-CD3 mAb at continuous time points was isolated and reversed to cDNA. The cytokine mRNA profiles of IL-2, IL-10 and IFN-γ were quantified by real-time PCR. Results A method of cytokine mRNA quantification was set up with specific primers and SYBR Green. The cytokine mRNA of IL-2, IL-10 and IFN-γ was up-regulated by the stimulation of anti-CD3 mAb in comparison with the controls. The content of IL-2 mRNA accumulated immediately and reached to the peak value of 14 000 copies/ml after the stimulation of anti-CD3 mAb for 12 h. The peak values of IL-10 and IFN-γ were 15 000 copies/ml and 110 000 copies/ml, respectively, after 24 to 48 hours＇ co-culture. Conclusion 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 immune response study.
出处
《江苏医药》
CAS
CSCD
北大核心
2007年第5期486-488,共3页
Jiangsu Medical Journal
基金
江苏大学临床医学技术发展基金(JLY20050055)
