siRNA沉默t-bet基因对CD4、CD8T淋巴细胞亚群IFN-γ产生的影响  

The effects of siRNA-mediated t-bet gene silencing on IFN-γ production of human CD4 and CD8 T lymphocyte subsets

作　　者：汪洪涛[1,2] 葛晓松[1] 李柏青[1,2] 

机构地区：[1]蚌埠医学院免疫学教研室 [2]安徽省感染与免疫重点实验室,蚌埠233003

出　　处：《生物学杂志》2009年第3期78-80,74,共4页Journal of Biology

基　　金：安徽省自然科学基金项目(050430603);蚌埠医学院科研项目(BY0826)资助

摘　　要：利用化学合成针对人t-bet基因的小干扰RNA(siRNA),转染体外培养的人外周血单个核细胞,利用流式细胞仪分选纯化CD4+、CD8+T淋巴细胞,半定量RT-PCR检测CD4+、CD8+T淋巴细胞中t-betmRNA的表达变化,流式细胞术检测转染前后IFN-γ产生的变化情况。探讨转录因子t-bet对人CD4+、CD8+T淋巴细胞亚群IFN-γ产生的调控作用。与对照组相比,转染后的人CD4+、CD8+T淋巴细胞t-betmRNA表达水平明显下降;转染siRNA后,CD4+T淋巴细胞中IFN-γ+细胞比例为(18.46±6.86)%,与对照组(50.20±5.91)%比较有显著性差异(P<0.01);CD8+T淋巴细胞IFN-γ+细胞比例为(74.18±9.33)%,和对照组(76.51±6.49)%比较差异不明显(P>0.05)。体外转录合成的siRNA可有效降低人CD4+、CD8+T淋巴细胞t-bet的基因表达;转录因子t-bet对人不同淋巴细胞亚群IFN-γ的产生所起作用不同。To investigate the effects of siRNA specificity for t-bet on IFN-γ production of human CD4^＋ and CD8^＋ T lymphocyte subsets activated by anti-CD3mAb, peripheral blood mononuclear cells （PBMC） from normal human subjects were stimulated with anti- CD3mAb to generate anti-CD3mAb activated T cells （CD3AT） which were almost αγT cells. CD4^ ＋ and CD8^＋ T lymphocytes by flow cytometry were sorted with anti-CD4^＋ or CD8^＋ monoclonal antibody from CD3AT. The expression of t-bet gene mRNA was detected by RT-PCR assay. The change of IFN-γ production was determined by flow cytometry method. After transfection, the mRNA expressions of t-bet gene in CD4^＋ and CD8^＋ T lymphocytes were decreased by using RT-PCR assay. Intracellular cytokine detection was assayed by flow cytometry. The IFN-γ^＋ cells in CD4^＋ T cells （50. 20% ± 5.91% ） were decreased in those transfected with t-bet siRNA （18.46% ±6. 86% ）, whereas the IFN-γ^＋ ceils in CD8^＋T ceils （74, 18 % ± 9. 33 % ） were unchanged in those compared with negative control groups （76. 51% ±6. 49% ）. Result showed that the RNA interference targeting t-bet could effectively inhibit the expression of t-bet mRNA. Different effects of levels of transcription factors on the regulation of IFN-γ production in CD4 ^＋ and CD8^＋T cells were made.

关 键 词：SIRNA T-BET IFN-Γ 淋巴细胞 

分 类 号：R392.11[医药卫生—免疫学]