氢醌对人骨髓单个核细胞拓扑异构酶Ⅱα表达的影响

Effect of hydroquinone on expression of topoisomerase enzyme Ⅱα in human bone marrow mononuclear cells

目的 研究苯代谢物氢醌（hydroquinone,HQ）对人骨髓单个核细胞中拓扑异构酶（TOPO）Ⅱα表达的影响,探讨TOPOⅡα在HQ所致造血毒性中的作用及调控机制.方法 50μmol/LHQ处理的正常人骨髓单个核细胞为实验组,设立对照组（等体积灭菌蒸馏水培养）,应用TOPOⅡ检测试剂盒检测TOPOⅡ的活力,免疫印迹法（Western blotting）检测TOPOⅡα含量的变化,反转录-聚合酶链反应（RT-PCR）法检测TOPOⅡα mRNA表达水平的改变,染色质免疫沉淀分析法（ChIp）检测TOPOⅡα含量的变化.结果 （1）正常人骨髓单个核细胞50 μmol/LHQ处理10 h后,TOPOⅡ的活力下降,TOPOⅡα含量以及TOPOⅡαmRNA水平分别为0.017±0.029、0.610±0.128,较对照组明显下降,差异有统计学意义（P〈0.01）;（2）TOPOⅡα含量降低伴随着TOPOⅡα启动子组蛋白H4乙酰化水平的明显降低（分别为1.198±0.056、0.324±0.229）,差异有统计学意义（P〈0.01）,不伴有组蛋白H3乙酰化水平的改变,分别为1.253±0.045、1.177±0.025;（3）HQ接触不同时间TOPOⅡαmRNA水平呈动态变化,且TOPOⅡα启动子水平的变化早于mRNA水平的变化.结论 HQ可抑制造血细胞TOPOⅡα的表达.组蛋白化学修饰水平的改变在苯代谢物所致的造血毒性中发挥一定的作用.

Objective To investigate the effects of hydroquinone （HQ） on expression of topoisomerase Ⅱα （TOPO Ⅱα ） in human bone marrow mononuclear cells,and to explore the role and possible regulatory mechanism of TOPO Ⅱα involved in toxicity of HQ to hematopoietic cells. Methods After human bone marrow mononuclear cells were exposed to 50 mol/L HQ （used the cells which were exposed to sterile distilled water as control）; the activity of TOPO Ⅱ was measured by TOPO Ⅱ assay kit; the expression levels of TOPO Ⅱα mRNA and protein were detected by RT-PCR technique and Western bloting method respectively;the chromatin immunoprecipitation （ChIP） assay was carried out to study the possible mechanism of TOPO Ⅱ α expression changes. Results （1）The activity of TOPO Ⅱ was inhibited obviously; the protein and mRNA expression of TOPO Ⅱα were 0.017±0.029 and 0.610±0.128, significantly lower than that in the control with the significant difference （P〈0.01） after treated with HQ for 10 h; （2） The decreased content of TOPO Ⅱα was associated with descented level of histone H4 acetylation than in the control, from 1.198±0.056 to 0.324±0.229, with the significant difference （P〈0.01）,without accompanied descented level of histone H3 acetylation, from 1.253±0.045 to 1.177±0.025 （P〉0.05）; （3） TOPO Ⅱα mRNA expression decreased gradually after HQ processing, and the chemical modification （histone H4 acetylation） of TOPO Ⅱα promoter happened prior to the mRNA expression. Conclusion HQ could repress the expression of TOPO Ⅱα in human bone marrow mononuclear cells; the change of histone chemical modification plays an important role in the benzene＇s hematopoietic toxicity.