CAJ | 학술논문

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.
목적 연구분대사물경곤(hydroquinone,HQ)대인골수단개핵세포중탁복이구매(TOPO)Ⅱα표체적영향,탐토TOPOⅡα재HQ소치조혈독성중적작용급조공궤제.방법 50μmol/LHQ처리적정상인골수단개핵세포위실험조,설립대조조(등체적멸균증류수배양),응용TOPOⅡ검측시제합검측TOPOⅡ적활력,면역인적법(Western blotting)검측TOPOⅡα함량적변화,반전록-취합매련반응(RT-PCR)법검측TOPOⅡα mRNA표체수평적개변,염색질면역침정분석법(ChIp)검측TOPOⅡα함량적변화.결과 (1)정상인골수단개핵세포50 μmol/LHQ처리10 h후,TOPOⅡ적활력하강,TOPOⅡα함량이급TOPOⅡαmRNA수평분별위0.017±0.029、0.610±0.128,교대조조명현하강,차이유통계학의의(P<0.01);(2)TOPOⅡα함량강저반수착TOPOⅡα계동자조단백H4을선화수평적명현강저(분별위1.198±0.056、0.324±0.229),차이유통계학의의(P<0.01),불반유조단백H3을선화수평적개변,분별위1.253±0.045、1.177±0.025;(3)HQ접촉불동시간TOPOⅡαmRNA수평정동태변화,차TOPOⅡα계동자수평적변화조우mRNA수평적변화.결론 HQ가억제조혈세포TOPOⅡα적표체.조단백화학수식수평적개변재분대사물소치적조혈독성중발휘일정적작용.
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.