论文部分内容阅读
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1×106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P15INK4B基因甲基化情况,实时荧光RT-PCR相对定量法检测P15INK4B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)P15INK4B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P15INK4B基因完全甲基化状态,在一定程度上抑制SKM-1细胞的增殖,使细胞分化阻滞在S期,同时促进细胞凋亡,并随DAC浓度的增加而增强;尚未发现DAC逆转P15INK4B基因甲基化状态的同时使沉默的P15INK4B基因mRNA重新表达。
Objective To investigate the effect of decitabine (DAC), a DNA methyltransferase inhibitor, on the methylation status of P15INK4B gene in myelodysplastic syndrome (MDS) leukemia cell line SKM-1. Methods SKM-1 cells in logarithmic growth phase were divided into 4 groups with 1 × 106 cells in each group. Among them, 3 groups were treated with DAC, SKM-1 cells were treated with 1.5, 3.0 and 6.0 μmol / L DAC for 48 h, respectively One group of SKM-1 cells without DAC were cultured for 48 h as a control group. Methylation-specific PCR (MSP) was used to detect the methylation status of P15INK4B gene in each group. The mRNA expression of P15INK4B was detected by real-time RT-PCR, and the fluorescence intensity of fluorescein diacetate succinimidyl ester (CFSE) The cell proliferation was measured by propidium iodide (PI) staining. The cell cycle was detected by PI staining and the early apoptosis was detected by Annexin V-FITC / PI double staining. Results 1) The methylation of P15INK4B gene was completely methylated in SKM-1 cells in control group. The methylation bands of three DAC-treated groups gradually became lighter and the unmethylated bands appeared and gradually deepened. 2) The expression of P15INK4B mRNA : The control group and the DAC with 1.5, 3.0, 6.0μmol / L of the three treatment groups were 1.00 ± 0.12 and 0.91 ± 0.13,0.51 ± 0.06,0.43 ± 0.04 (P <0.05), the differences between the three DAC treatment groups (P> 0.05) .3) The mean fluorescence intensity (MFI) of CFSE: 51.67 ± 1.61 and 55.33 ± 2.28,56.33 ± 2.50 respectively in the three treatment groups with 1.5, 3.0 and 6.0 μmol / L DAC, 55.71 ± 2.87, 3.0μmol / L group was more obvious than the control group (P <0.05), while there was no significant difference between each DAC treatment group (P> 0.05); 4) The proportion of cells in G1 phase The three treatment groups with DAC of 1.5, 3.0 and 6.0 μmol / L were 55.78 ± 3.61 and 48.12 ± 2.93, 51.69 ± 1.60 and 46.71 ± 1.54, respectively. The percentage of cells in S phase was 44.22 ± 3.61 and 51.88 ± 2.93, 48.31 ± 1.60 and 53.29 ± 1.54, respectively, while 1.5, 6.0μmol / L group showed more obvious changes than the control group (P <0.05) With significant difference (P <0.05); 5) early apoptosis rate (%): control group and DAC The three treatment groups with 1.5, 3.0 and 6.0 μmol / L were 2.91 ± 0.26 and 7.77 ± 0.22, 12.45 ± 0.28 and 13.86 ± 0.27, respectively (P <0.05). There was no significant change in each treatment group (P <0.05). Conclusion DAC can reverse the complete methylation status of P15INK4B gene in SKM-1 cells and inhibit the proliferation of SKM-1 cells to a certain extent, and block the differentiation of cells in S phase and promote the apoptosis of SKM-1 cells. With the increase of DAC concentration, But did not find that DAC reverses the methylation status of P15INK4B gene and re-express silenced P15INK4B mRNA.