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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RPMI8226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0-5μmol/L5-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Graphpad5.0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RPMI8226细胞株XAF1基因启动子CpG岛均存在过甲基化。XG-7和RPMI8226细胞株经2.5μmol/L5-氮杂胞苷处理72小时后仅表达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-氮杂胞苷处理48小时抑制XG-7骨髓瘤细胞株的IC50值为2.6μmol/L。1.0、2.0、2.5、5.0μmol/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)%。1.0-4.0μmol/L5-氮杂胞苷与1.0-4.0μmol/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。
This study was to investigate the effect of 5-azacytidine on the expression of XAF1 gene in myeloma cell lines and the proliferation of myeloma cells in vitro. The expression of XAF1 gene in myeloma cell lines RPMI8226 and XG-7 was detected by RT-PCR. Methylation status of CpG island of XAF1 gene was detected by methylation-specific PCR (MSP). Myeloma cell lines were treated with 0-5 μmol / L 5-azacytidine. The inhibitory effect of 5-azacytidine on the proliferation of myeloma cells was detected by CCK-8 colorimetric assay. The growth inhibitory effect of 5-azacytidine on myeloma cells was analyzed by Graphpad5.0 software. Apoptosis was detected by Annexin V / 7-AAD staining. The results showed that: XG-7 cells do not express XAF1 mRNA, and RPMI8226 cells express XAF1 mRNA transcripts 1 and 2. XG-7 and RPMI8226 cell line XAF1 gene promoter CpG islands are hypermethylated. XG-7 and RPMI8226 cell lines only expressed XAF1 mRNA transcript 1 after 72 hours treatment with 2.5 μmol / L 5-azacytidine, and the methylation of CpG island of XAF1 gene promoter was decreased. 5-Azacytidine anti-myeloma effect in a time and concentration-dependent manner. 48h treatment with 5-azacytidine inhibited the IC50 value of XG-7 myeloma cell line to 2.6μmol / L. The apoptotic rates of XG-7 cells treated with 1.0,2.0,2.5,5.0μmol / L 5-azacytidine for 48 hours were (34.3 ± 8.0)%, (54.8 ± 3.1)%, (64.1 ± 3.4)%, (81.0 ± 4.1)%. The combination of 1.0-4.0μmol / L 5-azacytidine with 1.0-4.0μmol / L arsenic trioxide has a synergistic anti-myeloma effect with the combined index of less than 1.0. Conclusion: The lack of XAF1 expression in myeloma cells is related to the over-methylation of CpG island. 5-azacytidine has an anti-myeloma effect at a clinically achievable drug concentration and its mechanism of action is to induce apoptosis of myeloma cells in synergistic anti-myeloma effect with arsenite.