中文摘要：

目的探讨DNA甲基转移酶抑制剂地西他滨（DAC）对骨髓增生异常综合征（MDS）转白血病细胞株SKM-1 P15INK4B基因甲基化状态的影响。方法对数生长期的SKM-1细胞设4组,每组1×10^6个细胞,其中3组为DAC处理组,分别以1.5、3.0和6.0μmol/L DAC处理SKM-1细胞48 h,另1组未加DAC的SKM-1细胞培养48 h作为对照组。甲基化特异性PCR（MSP）检测各组细胞P15^INK4B基因甲基化情况,实时荧光RT-PCR相对定量法检测P15^INK4B基因mRNA表达情况,羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）法检测细胞增殖抑制情况,碘化丙啶（PI）单染法检测细胞周期,Annexin V-FITC/PI双染法检测细胞早期凋亡情况。结果 1）对照组SKM-1细胞的P15INK4B基因完全甲基化,3个DAC处理组的甲基化条带逐渐变浅,非甲基化条带出现并逐步加深;2）P15^INK4B基因mRNA表达水平：对照组与DAC为1.5、3.0、6.0μmol/L的3个处理组分别为1.00±0.12与0.91±0.13、0.51±0.06、0.43±0.04（P〈0.05）,3个DAC处理组之间差异甚微（P〉0.05）;3）CFSE平均荧光强度（MFI）：对照组与DAC为1.5、3.0、6.0μmol/L的3个处理组分别为51.67±1.61与55.33±2.28、56.33±2.50、55.71±2.87,仅3.0μmol/L组较对照组变化较明显（P〈0.05）,而各DAC处理组之间变化差异很小（P〉0.05）;4）G1期细胞比例（%）：对照组与DAC为1.5、3.0、6.0μmol/L的3个处理组分别为55.78±3.61与48.12±2.93、51.69±1.60、46.71±1.54,S期细胞比例（%）：4个组分别为44.22±3.61与51.88±2.93、48.31±1.60、53.29±1.54,其中1.5、6.0μmol/L组较对照组变化较明显（P〈0.05）,而DAC各处理组中,6.0与3.0μmol/L组之间具明显差异（P〈0.05）;5）早期凋亡率（%）：对照组与DAC为1.5、3.0、6.0μmol/L的3个处理组分别为2.91±0.26与7.77±0.22、12.45±0.28、13.86±0.27（P〈0.05）,DAC各处理组没有明显变化（P〈0.05）。结论 DAC能逆转SKM-1细胞的P15^INK4B基因完全甲基化状态,在一定程度上?

英文摘要：

Objective To explore the demethylation effect of P15INK4 Bgene in myelodysplastic syndrome（ MDS）-derived cell line SKM-1 induced by DNA methyltransferase inhibitor Decitabine（ DAC）. Methods SKM-1 cells in exponential growth phase were set up for 4 groups. 3 DAC groups of SKM-1 cells were treated with 1. 5,3. 0 and 6. 0 mol / L DAC for48 h,untreated SKM-1 cells were the control group. The methylation of P15INK4 Bgene,the expression of P15INK4 Bgene mRNA,the cellular proliferation,the cell cycle and the cellular early apoptosis were detected by methylation specific PCR（ MSP）,real time fluorescent RT-PCR,CFSE methods,propidium iodide（ PI） staining and annexin V-FITC/PI double la-beling. Results（ 1） The control group was entirely methylated with P15^INK4 Bgene,while 1. 5 mol / L,3. 0 mol / L and 6. 0 mol / L DAC groups underwent partial methylation and showed a unmethylated and darkening strip. The degree of P15INK4 B gene meth-ylation was lower in the groups with higher concentrations of DAC.（ 2） The expression of P15^INK4 Bgene mRNA in the control group,1. 5 mol / L,3. 0 mol / L and 6. 0 mol / L DAC groups were 1. 00 ± 0. 12,0. 91 ± 0. 13,0. 51 ± 0. 06 and 0. 43 ± 0. 04,respectively. Compared with control group,the expression of P15 I INK4B gene mRNA in DAC groups significantly decreased（ P 〈0. 05）. There were no statistical significant differences when compared with DAC groups（ P 〉0. 05）.（ 3） The MFI of CFSE in the control group,1. 5 mol / L,3. 0 mol / L and 6. 0 mol / L DAC groups were 51. 67 ± 1. 61,55. 33 ± 2. 28,56. 33 ±2. 50 and 55. 71 ± 2. 87,respectively. Compared with control group,the 3. 0 mol / L DAC group significantly decreased（ P 〈0. 05）. There were no statistical significances when compared with DAC groups（ P 〉0. 05）. 4）. The proportion of G1 phase cells in the control group,1. 5 mol / L,3. 0 mol / L and 6. 0 mol / L DAC groups were（ 55. 78 ± 3. 61） %,（ 48. 12 ±2. 93） %,（ 51. 69 ± 1. 60） % and（ 46. 71 ± 1. 54） %,respect