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目的探讨LSD1的脱甲基酶活性对人外周血CD4+T细胞Th1/Th2分化格局的影响及其分子机制。方法 anti-CD3/28刺激活化人外周血CD4+T细胞48 h后,shLSD1和反苯环丙胺(TCP)抑制LSD1的表达,流式细胞术检测细胞内细胞因子IFN-γ、IL-4的表达情况;逆转录聚合酶链式反应(RT-PCR)和实时荧光定量PCR(RQ-PCR)检测不同浓度TCP对T细胞IFN-γ、IL-4、Tbet mRNA表达的影响;Western blot观察LSD1、T-bet、STAT1和pSTAT1蛋白表达情况。结果 shLSD1和TCP组细胞IFN-γ+T细胞比例[(26.13±1.89)%和(27.01±1.18)%]明显高于对照组[(14.67±0.65)%,(P<0.05)],而IL-4+T细胞比例与对照组无显著变化(P>0.05);RT-PCR检测显示,shLSD1和TCP组IFN-γmRNA表达明显高于对照组(P<0.05),IL-4基因表达则没有改变(P>0.05);转染shLSD1或TCP处理引起T-bet、pSTAT1表达明显高于对照组(P<0.05),而STAT1表达无明显变化(P<0.05)。结论下调LSD1表达可以促进人CD4+T细胞向Th1方向分化,对Th2方向细胞无影响。其分子机制涉及T-bet/STAT1信号通路。
Objective To investigate the effect of demethylase activity of LSD1 on the Th1 / Th2 differentiation pattern of human peripheral blood CD4 + T cells and its molecular mechanism. Methods After anti-CD3 / 28 stimulation of human peripheral blood CD4 + T cells for 48 h, the expression of LSD1 was inhibited by shLSD1 and tranylcypromine (TCP). The levels of cytokines IFN-γ and IL-4 were detected by flow cytometry The expression of IFN-γ, IL-4 and Tbet mRNA in T cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative PCR (RQ-PCR) , T-bet, STAT1 and pSTAT1 protein expression. Results The proportion of IFN-γ + T cells in shLSD1 and TCP groups was significantly higher than that in control group [(26.13 ± 1.89)% vs (27.01 ± 1.18)% vs (14.67 ± 0.65)%, P <0.05) (P> 0.05). The results of RT-PCR showed that the expression of IFN-γmRNA in shLSD1 and TCP groups was significantly higher than that in control group (P <0.05), while the expression of IL-4 gene was not (P> 0.05). The expression of T-bet and pSTAT1 in transfected shLSD1 or TCP group was significantly higher than that in control group (P <0.05), while STAT1 expression had no significant change (P <0.05). Conclusion Down-regulation of LSD1 expression can promote the differentiation of human CD4 + T cells into Th1 cells and has no effect on Th2 cells. Its molecular mechanism involves the T-bet / STAT1 signaling pathway.