CAJ | 학술논문

本研究探讨5-氮杂胞苷对骨髓瘤细胞株中XAF1基因表达的影响及体外抗骨髓瘤细胞增殖效率.采用逆转录PCR方法检测骨髓瘤细胞株RPMI 8226和XG-7中XAF1基因的表达.采用甲基化特异性PCR(MSP)方法检测XAF1基因CpG岛甲基化状态.采用0-5 μmol/L 5-氮杂胞苷处理骨髓瘤细胞株.采用CCK-8比色法检测5-氮杂胞苷处理对骨髓瘤细胞增殖抑制作用,应用Graphpad 5.0软件分析5-氮杂胞苷对骨髓瘤细胞的生长抑制作用.采用Annexin V/7-AAD染色流式细胞仪检测细胞凋亡.结果表明:XG-7细胞不表达XAF1 mRNA,RPMI8226细胞表达XAF1 mRNA转录本1和2.XG-7和RPMI 8226细胞株XAF1基因启动子CpG岛均存在过甲基化.XG-7和RPMI 8226细胞株经2.5 μmol/L 5-氮杂胞苷处理72 d、时后仅表达XAF1 mRNA转录本1,并且XAF1基因启动子CpG岛甲基化程度降低.5-氮杂胞苷抗骨髓瘤作用呈时间和浓度依赖性.5-氯杂胞苷处理48小时抑制XG-7骨髓瘤细胞株的IC50值为2.6 μmol/L.1.0、2.0、2.5、5.0 μmol/L浓度的5-氮杂胞苷处理XG-7细胞48小时后诱导细胞凋亡率分别为(34.3±8.0)%,(54.8±3.1)%,(64.1±3.4)%,(81.0±4.1)%.1.0-4.0 μmol/L5-氮杂胞苷与1.0-4.0 μmol/L亚砷酸联合应用具有协同抗骨髓瘤细胞作用,联合指数均小于1.0.结论:骨髓瘤细胞中XAF1表达缺失与启动子CpG岛过甲基化有关.5-氮杂胞苷在临床上能达到的药物浓度下具有抗骨髓瘤作用,其作用机制是诱导骨髓瘤细胞凋亡,与亚砷酸具有协同抗骨髓瘤作用.
본연구탐토5-담잡포감대골수류세포주중XAF1기인표체적영향급체외항골수류세포증식효솔.채용역전록PCR방법검측골수류세포주RPMI 8226화XG-7중XAF1기인적표체.채용갑기화특이성PCR(MSP)방법검측XAF1기인CpG도갑기화상태.채용0-5 μmol/L 5-담잡포감처리골수류세포주.채용CCK-8비색법검측5-담잡포감처리대골수류세포증식억제작용,응용Graphpad 5.0연건분석5-담잡포감대골수류세포적생장억제작용.채용Annexin V/7-AAD염색류식세포의검측세포조망.결과표명:XG-7세포불표체XAF1 mRNA,RPMI8226세포표체XAF1 mRNA전록본1화2.XG-7화RPMI 8226세포주XAF1기인계동자CpG도균존재과갑기화.XG-7화RPMI 8226세포주경2.5 μmol/L 5-담잡포감처리72 d、시후부표체XAF1 mRNA전록본1,병차XAF1기인계동자CpG도갑기화정도강저.5-담잡포감항골수류작용정시간화농도의뢰성.5-록잡포감처리48소시억제XG-7골수류세포주적IC50치위2.6 μmol/L.1.0、2.0、2.5、5.0 μmol/L농도적5-담잡포감처리XG-7세포48소시후유도세포조망솔분별위(34.3±8.0)%,(54.8±3.1)%,(64.1±3.4)%,(81.0±4.1)%.1.0-4.0 μmol/L5-담잡포감여1.0-4.0 μmol/L아신산연합응용구유협동항골수류세포작용,연합지수균소우1.0.결론:골수류세포중XAF1표체결실여계동자CpG도과갑기화유관.5-담잡포감재림상상능체도적약물농도하구유항골수류작용,기작용궤제시유도골수류세포조망,여아신산구유협동항골수류작용.
This study was aimed to investigate the effect of 5-azacytidine(5-AZA)on XAF1 expression in myeloma cells and efficacy of 5-AZA treatment for myeloma in vitro.XAFl expression was analyzed by semi-quantitative PCR.Methylation-specific PCR(MSP)was used to detect the methylation status of XAF1 promoter CpG islands.RPMI 8226and XG-7 cells were treated with 0-5 μmol/L of 5-AZA.Expression of XAF1 mRNA variants was confirmed by gel electrophoresis.The results indicated that the untreated RPMI 8226 cell expressed XAF1 mRNA transcript 1 and transcript 2,untreated XG-7 cells did not express XAF1 mRNA.Hypermethylation of XAF1 promoter CpG islands could be detected in both cell lines.Both cell lines expressed full-length XAF1 transcript after being treated with 2.5 μmol/L of 5-AZA for 72 hours.5-AZA treatment led XAF1 promoter CpG island to hypomethylation in both cell lines.5-AZA exerted anti-myeloma activity in a time-and concentration-dependent manner.The IC50 value of XG-7 cells treated with 5-AZA for 48 hours was 2.6 μmol/L.1.0,2.0,2.5 and 5.0 μmol/L of 5-AZA treatment for 48 hours induced(34.3±8.0)%,(54.8±3.1)%,(64.1±3.4)%,(81.0±4.1)%apoptosis in XG-7 cell line respectively.The combination of 1.0-4.0μmol/L of 5-AZA with 1.0-4.0 μmol/L of arsenic trioxide(ATO)exhibited synergistic toxicity in myeloma cells with all CI values less than 1.0.It is concluded that lack of XAF1 expression and abnormal expression of XAF1 in myeioma cell lines are associated with the hypermethylation of XAF1 gene promoter CpG island.5-AZA treatment can induce the expression of XAF1 mRNA and protein in myeloma.5-AZA exerts anti-myeloma activity via apoptosis at clinically achievable concentrations.The findings suggested that 5-AZA and ATO may be an effective combination in the therapy of patients with multiple myeloma.