胸腺肽α1调节AML细胞株IDO表达对T细胞免疫抑制作用影响的研究

Experimental research on the regulation of acute myeloid leukemia cells through indoleamine2,3-dioxygenase expression by thymosin-α1 for improving the immunosuppression mediated by T cells

目的吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)是色氨酸代谢过程中的限速酶,近年来研究发现,IDO高表达是急性髓系白血病(acute myeloid leukemia,AML)细胞介导免疫耐受的重要机制,因此研究如何调控IDO表达,将有可能为AML治疗提供新的辅助方法。本实验拟从分子生物及蛋白质水平,探讨胸腺肽α1(thymosin-α1,Tα1)调节AML细胞株IDO表达及改善T细胞的免疫抑制作用,为AML免疫治疗提供理论依据。方法采用逆转录-聚合酶联反应(RT-PCR)检测γ-干扰素(interferon-γ,IFN-γ)、Tα1对HL-60/K562细胞株IDO mRNA表达的影响;采用蛋白质印迹法检测IFN-γ和Tα1对HL-60/K562细胞株IDO蛋白表达的影响;采用MTT检测Tα1和1-甲基色氨酸(1-methyl-tryptophan,1-MT)作用后经IFN-γ诱导的HL-60/K562细胞对植物血凝素(phytohaemagglutinin,PHA)刺激T细胞增殖抑制作用。结果HL-60/K562细胞株均表达IDO mRNA;IFN-γ作用后,IDO mRNA表达明显增加(HL-60细胞株t=130.19,K562细胞株t=67.38,均P<0.05);IFN-γ+Tα1共同作用后,明显下调IDO mRNA表达(HL-60细胞株F=132.55,K562细胞株F=77.36,均P<0.05),并呈浓度依赖性(HL-60细胞株r=-0.71,K562细胞株r=-0.80);HL-60/K562细胞株低水平表达IDO蛋白;经IFN-γ作用后,HL-60细胞株IDO蛋白表达水平为2.505 6±0.019 6,明显高于空白对照组0.189 7±0.083 4,t=326.79,P<0.05;K562细胞株IDO蛋白表达水平为0.988 8±0.023 9,明显高于空白对照组0.253 6±0.016 3,t=76.12,P<0.05;IFN-γ+Tα1共同作用后,经IFN-γ+Tα1共同作用后,IDO蛋白在HL-60细胞株各组的表达水平分别为2.455 0±0.066 1、1.526 0±0.017 3、1.134 1±0.060 1、0.806 0±0.029 7和0.070 0±0.003 6,并呈浓度依赖性,F=4 187.76,r=-0.77,P<0.05;经IFN-γ+Tα1共同作用后,IDO蛋白在K562细胞株各组的表达水平分别为0.998 6±0.016 4、0.784 3±0.014 9、0.714 5±0.009 5、0.656 6±0.007 6和0.492 2±0.010 9,并呈浓度依赖性,F=59.50,r=-0.76,P<0.05。IFN-γ诱导的HL-60/K562细胞可抑制PHA刺激的T细胞增殖(P<0.05),并呈浓度依赖性(HL-60细胞株r=0.99,K562细胞株r=0.98);1-MT组和Tα1组中HL-60/K562细胞株对T细胞增殖的抑制率明显低于对照组,差异有统计学意义,P<0.05。结论 IDO可能在AML诱导机体产生免疫耐受过程中起重要作用,Tα1可下调AML细胞IDO表达,改善AML细胞对T细胞的免疫抑制作用。

OBJECTIVE Indoleamine2,3-dioxygenase （IDO） was a rate-limiting enzyme in the process of trypto- phan metabolism. Recently, researches had confirmed that high level of IDO expression was an important mechanism of acute myeloid leukemia （AML） cell-mediated immune tolerance. So studies how to control IDO expression may provide a new aiding method for treatment of AML. This study was to investigate the regulation of AML cells through IDO expres sion by thymosin α1 （Tα1）. It could improve the immunosuppression mediated by T ceils from both the level of molecular biology and protein. METHODS Expressions of IDO mRNA in HL 60/K562 cells cocultured with interferon-γ（IFN-γ） and Tα1 were determined by reverse transcription-polymerase chain reaction （RT-PCR）. The expression of IDO protein in HL-60/K562 cells cocultured with IFN-α and Tα1 was assayed by Western Blot. Inhibition of phytohaemagglutinin （PHA） activated T cells proliferation by IFN-γ-induced HL-60/K562 cells cocultured with Tα1 and 1-methyl-tryptophan （1 MT） was detected by MTT. RESUTLS HL-60/K562 cells were all expressed IDO. IFN -γ increased the expression of IDOmRNA（HL-60 cellst--130.19,K562 cellst=67.38,P〈0.05）. The expression of IDOmRNAinHL-60/K562 cells cocultured with IFN-γ＋Tα1 was decreased in concentration-dependent manner（HL-60 cells F= 132.55 ,r=-0.71. K562 cells F=77.36,r=-0.80;P〈0.05）. IDO protein was expressed at lower level in HL-60/K562 cells. IFN-T increased the expression of IDO protein（HL 60 cells t=326.79,K562 cells t=76.12,P〈0.05）. The expression of IDO protein in HL 60/K562 cells cocultured with IFN-γ＋ Tα1 was decreased with concentration dependent manner（HL-60 cells F=4 187.76,r=-0.77. K562 cellsF=59.50,r=-0.76, P〈0.05）. PHA-activatedTcells proliferation was inhibited by IFN-γ induced HL-60/K562 cells in a concentration-dependent way（HL-60 cells r=0.99,K562 cells r=0.98,P〈0.05） ; The inhibition rates were significantly decreased in HL-60/K562 cells cocultured with Tα1 and 1-MT（P〈0.05）. CON- CLUSIONS IDO may play an important role in immune tolerance induced by leukemia cells. The immunosuppression me- diated by leukemia cells could be improved by Tα1 through dowwregulation of IDO expression.