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目的:研究曲古抑菌素A(Trichostatin A,TSA)下调γ干扰素(interferon-gamma,IFN-γ)诱导的人肝癌细胞HepG2内吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)表达的分子机制。方法:Western blot检测TSA在IFN-γ诱导的HepG2细胞中IDO的表达、信号转导及转录激活子1(STAT1)的磷酸化和干扰素调节因子1(IRF-1)的诱导表达情况。用免疫细胞化学法检测TSA处理HepG2细胞后对IDO表达的影响。流式细胞术分析TSA处理后IFN-γ受体2表达量的变化,进一步在激光共聚焦显微镜下观察TSA对STAT1核转位的影响,利用双荧光素酶报告基因系统检测TSA对IFN-γ激活位点(γ-activated sites,GAS)、干扰素刺激应答元件(interferonstimulated response elements,ISRE)和核因子-κB(NF-κB)的激活的影响。结果:TSA以剂量依赖方式下调HepG2细胞内IFN-γ诱导的IDO表达、能明显抑制STAT1第701位酪氨酸的磷酸化和STAT1的核转位,但是上调IFN-γ受体2受体的表达。双荧光素酶报告基因分析和Westernblot结果表明:TSA能显著抑制GAS和IRF-1的激活却不能抑制NF-κB和ISRE的激活。结论:TSA能下调IFN-γ诱导的HepG2细胞中IDO的表达,其机制可能是与其抑制STAT1的磷酸化和核转位,以及抑制STAT1与GAS的结合有关,而不是通过下调IFN-γ受体的表达来实现的。
Objective: To study the effects of trichostatin A (TSA) on the expression of indoleamine 2,3-dioxygenase in HepG2 cells induced by interferon-gamma (IFN-γ) 3-dioxygenase, IDO). Methods: Western blot was used to detect the expression of IDO, the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and the induction of interferon regulatory factor 1 (IRF-1) in HepG2 cells induced by IFN-γ. Immunocytochemistry was used to detect the effect of TSA on IDO expression in HepG2 cells. Flow cytometry was used to analyze the change of IFN-γreceptor 2 expression after TSA treatment. The effect of TSA on STAT1 nuclear translocation was observed by laser scanning confocal microscopy. The dual luciferase reporter gene system was used to detect the effect of TSA on IFN-γ (GAS), the activation of interferon-stimulated response elements (ISRE) and nuclear factor-κB (NF-κB). Results: TSA down-regulated the expression of IDO induced by IFN-γ in HepG2 cells in a dose-dependent manner, and significantly inhibited tyrosine phosphorylation of STAT1 at position 701 and nuclear translocation of STAT1, but up-regulated the expression of IFN-γ receptor 2 expression. Dual luciferase reporter assay and Western blot showed that TSA can significantly inhibit the activation of GAS and IRF-1 but not the activation of NF-κB and ISRE. CONCLUSIONS: TSA can down-regulate the expression of IDO in HepG2 cells induced by IFN-γ, which may be related to its inhibition of phosphorylation and nuclear translocation of STAT1 and the binding of STAT1 to GAS, not down-regulation of IFN-γ receptor To achieve the expression.