ESAT-6刺激结核患者CD4^+ T淋巴细胞前后释放IFN-γ mRNA差异表达及信号通路变化

Changes of release IFN-γ mRNAs expression profiling and signaling pathways of ESAT-6 stimulated CD4^+ T lymphocytes from tuberculosis patients

目的利用全基因组表达谱芯片对早期分泌靶向抗原6(ESAT-6)刺激结核患者外周血CD4^+T淋巴细胞前后释放γ-干扰素(IFN-γ)mRNA进行检测,并对mRNA在刺激过程中可能涉及到的信号通路变化进行分析。方法抽取3例初诊结核患者外周静脉血各10 ml,密度梯度离心法分离获得外周血单个核细胞(PBMC),ESAT-6刺激PBMC,6 h后磁珠法分选CD4^+T淋巴细胞,提取细胞总RNA,全基因组表达谱芯片检测刺激前后差异表达基因。利用GO分析对差异表达的基因进行功能分类,KEGG进行信号通路分析。结果 ESAT-6刺激后差异表达的mRNA共有2 297条,相对高表达的mRNA有1 071条,相对低表达的mRNA有1 226条(>2倍变化且P<0.05)。信号通路分析显示差异表达的mRNA参与了17条信号通路的调控,其中JAK-STAT信号通路、Toll样受体信号通路、NF-κB信号通路、MAPK信号通路、PI3K-Akt信号通路等5条信号通路与结核释放IFN-γ关系密切。结论 ESAT-6刺激后的CD4^+T淋巴细胞有大量mRNA发生差异表达,其在多个与结核释放IFN-γ密切相关的信号通路中发挥着重要的调控作用。

Objective To detect the difference in release IFN-γ mRNA expression profiling of early secreted antigenic target 6 ku protein（ ESAT-6） stimulated CD4^＋T lymphocytes from tuberculosis patients peripheral blood and also to identify the changes in signaling pathways through whole genome expression microarray screening. Methods10 ml peripheral venous blood was collected from three first diagnosed tuberculosis patients. Peripheral blood mononuclear cell（ PBMC） was isolated by density gradient centrifugation and stimulated with ESAT-6 for 6 hours.Then,CD4~＋T lymphocytes were obtained from ESAT-6 stimulated PBMC by immunomagnetic technique. Total RNA was extracted,useing whole genome expression microarray to detect differences in gene expression before and after stimulation. GO and KEGG were used to analyze the functional classification of differentially expressed genes and signaling pathways. Results After ESAT-6 stimulation,it had 2 297 differentially expressed mRNAs. 1 071 mRNAs were up-regulated,1 226 mRNAs were down-regulated（ 2 fold changes and P〈0. 05）. 17 signaling pathways were involved based on mRNAs expression data and 5 of which were involved in tuberculosis IFN-γ release（ JAK-STAT signaling pathway,toll like receptor signaling pathway,NF-κB signaling pathway,MAPK signaling pathway,and PI3K-Akt signaling pathway）. Conclusion After ESAT-6 stimulation,obvious changes of mRNAs expression profiling are observed. Those differentially expressed mRNAs play important roles in regulating signaling pathways in CD4^＋T lymphocytes tuberculosis IFN-γ release.