CAJ | 학술논문

目的探讨慢性髓性白血病细胞系K562细胞甲基化诱导静止基因(TMS1)甲基化程度及三氧化二砷(As2O3)对其甲基化状态的逆转作用及其机制.方法 K562细胞经不同浓度As2O3处理48 h,采用甲基化特异性PCR(MSP)法检测As2O3处理前后K562细胞TMS1基因甲基化程度;RT-PCR及Western blot法检测As2O3处理前后K562细胞TMS1 mRNA及蛋白表达,Western blot法检测凋亡蛋白Bcl-2、Bax表达水平的变化;用细胞凋亡检测试剂盒(Annexin V/PI)标记、流式细胞术检测细胞凋亡.结果 K562细胞TMS1基因呈完全甲基化,TMS1 mRNA及蛋白呈低表达状态,相对表达水平分别为0.01±0.01、0.09±0.02;2 μtmol/L As2O3可完全逆转K562细胞TMS1的甲基化水平,TMSl mRNA及蛋白相对表达水平分别为0.72±0.04和1.3±0.06,与处理前相比明显升高(P＜0.01);细胞凋亡检测结果显示,与对照组相比,2 μmol/L As2O3处理组细胞凋亡率明显增加[(2.05±0.16)％对(12.24±1.06)％,P＜0.05)].与对照组相比,2μmol/L As2O3处理组K562细胞抗凋亡蛋白Bcl-2表达降低,促凋亡蛋白Bax表达明显增高,Bcl-2/Bax比值明显降低(1.94±0.14对0.56±0.12,P＜0.01).结论 As2O3可通过逆转抑癌基因TMS1的甲基化状态,恢复其表达,并通过下调Bcl-2/Bax比值促进K562细胞凋亡.
목적 탐토만성수성백혈병세포계K562세포갑기화유도정지기인(TMS1)갑기화정도급삼양화이신(As2O3)대기갑기화상태적역전작용급기궤제.방법 K562세포경불동농도As2O3처리48 h,채용갑기화특이성PCR(MSP)법검측As2O3처리전후K562세포TMS1기인갑기화정도;RT-PCR급Western blot법검측As2O3처리전후K562세포TMS1 mRNA급단백표체,Western blot법검측조망단백Bcl-2、Bax표체수평적변화;용세포조망검측시제합(Annexin V/PI)표기、류식세포술검측세포조망.결과 K562세포TMS1기인정완전갑기화,TMS1 mRNA급단백정저표체상태,상대표체수평분별위0.01±0.01、0.09±0.02;2 μtmol/L As2O3가완전역전K562세포TMS1적갑기화수평,TMSl mRNA급단백상대표체수평분별위0.72±0.04화1.3±0.06,여처리전상비명현승고(P＜0.01);세포조망검측결과현시,여대조조상비,2 μmol/L As2O3처리조세포조망솔명현증가[(2.05±0.16)％대(12.24±1.06)％,P＜0.05)].여대조조상비,2μmol/L As2O3처리조K562세포항조망단백Bcl-2표체강저,촉조망단백Bax표체명현증고,Bcl-2/Bax비치명현강저(1.94±0.14대0.56±0.12,P＜0.01).결론 As2O3가통과역전억암기인TMS1적갑기화상태,회복기표체,병통과하조Bcl-2/Bax비치촉진K562세포조망.
Objective To detect the methylation status of TMS1 gene and its demethylation by arsenic trioxide (As2O3) in K562 cells.Methods K562 cells were treated with different concentrations of As2O3 for 48 hours.Methylation-specific PCR (MSP) was used to determine the methylation status of TMS1.RT-PCR and Western blot were used to detect the levels of TMS1 mRNA and protein.TMS1 associated apoptosis proteins Bcl-2/Bax were also analyzed by Western blot.Apoptosis were evaluated by flow cytometry using Annexin V/propium iodide (PI) double staining.Results TMS1 gene was completely methylated in K562 cells and the levels of TMS1 mRNA and protein were low (0.01 ±0.01,0.09±0.02),which could be reversed (mRNA:0.72±0.04; protein:1.30±0.06; P＜0.01) by 2 μmol/L As2O3 via overt demethylation of TMS1 gene.Apoptosis in experiment group (12.24 ± 1.06) was significantly higher than that in control group (2.05 ± 0.16,P＜0.05).In experiment group,the down-expression of antiapoptotic protein Bcl-2 and up-expression of pro-apoptotic protein Bax led to an obvious decline ratio of Bcl-2/Bax (0.56 ± 0.12),as compared to the control group (1.94 ± 0.14,P＜0.01).Conclusion As2O3 could up-regulate TMS1 gene expression by reversing its hypermethylation and induced apoptosis by downregulation of Bcl-2/Bax ratio in K562 cells.