【摘要】 目的以骨髓增生异常综合征(myelodysplastic syndromes,MDS)细胞系SKM-1细胞为研究对象,在体外观察青蒿琥酯(artesunate,ART)对SKM-1细胞脱氧核糖核酸甲基转移酶(DNA methyltransferase,DNMT1)基因表达的影响,以期为临床应用ART治疗中高危MDS患者提供体外实验的理论依据。方法 ART干预体外细胞培养MDS细胞SKM-1细胞系,采用逆转录聚合酶链反应(revers transcription-polymerase chain reaction,RTPCR)法观察DNMT1的mRNA改变,采用蛋白印迹法观察DNMT1蛋白表达改变。采用实时定量RT-PCR法观察DNMT1靶基因p15INK4b表达的改变,采用甲基化PCR法观察p15INK4b启动子甲基化的改变。结果 1、5、10μmol/L的ART处理SKM-1细胞后DNMT1基因表达低于对照组,差异有统计学意义(P<0.05)。1、5、10μmol/L各剂量组间差异无统计学意义(P>0.05)。DNMT1蛋白表达1、10μmol/L低于对照组,差异有统计学意义(P<0.05);1、10μmol/L组低于5μmol/L组,差异有统计学意义(P<0.05);ART处理SKM-1细胞后DNMT1基因3、6、12h处理组低于对照组,差异有统计学意义(P<0.05);3h、6h与12h差异有统计学意义(P<0.05)。DNMT1蛋白3、6、12h处理组低于对照组,差异有统计学意义(P<0.05)。ART处理后p15INK4b的mRNA水平5、10μmol/L组、地西他宾组高于对照组,差异有统计学意义(P<0.05)。p15INK4b的mRNA水平在10μmol/L组是这5个处理组中最高的,并且1μmol/L、5μmol/L、地西他宾组与10μmol/L组比较,差异有统计学意义(P<0.05);ART处理SKM-1细胞后DNMT1蛋白靶基因p15INK4b启动子甲基化程度下降,各组差异无统计学意义;ART组非甲基化启动子较其他2组上升,差异有统计学意义(P<0.05)。结论ART通过抑制DNMT1的转录和蛋白表达来恢复SKM-1细胞中的p15INK4b的表达,进而发挥其抑癌作用。更多还原

【Abstract】 Objective To observe the effects of artesunate(ART)on gene expression levels of DNMT1 by using SKM-1cells as the research object,and provide laboratory evidence in vitro for the usage of ART in MDS treatment.Methods After being treated with ART(1,5and 10μmol/L),DNMT1 mRNA changes in SKM-1cells were observed with RT-PCR,and the protein expression changes were observed with West Blot.Then,the expression changes of p15INK4 b,the DNMT1 targeting gene were observed by using real-time quantitative RT-PCR,and the changes of p15INK4 bpromoter methylation state were observed by using methylation PCR.Results After being treated with ART(1,5and 10μmol/L),the difference was statistically significant for DNMT1 gene expression in SKM-1cells compared with control group(P<0.05),but there was no significant difference in the three dose groups(1,5and 10μmol/L)(P>0.05).As to DNMT1 protein expression,compared with 5μmol/L group,there was significantly lower in the other two groups(1and 10μmol/L)(P<0.05),and also significantly lower than that in control group(P<0.05).However there was no significant difference among the other groups(P>0.05).After SKM-1cells being treated with ART for 3h,6hand 12 h,compared with control group,the difference of DNMT1 gene and protein expressions in the three groups(3h,6h and 12h)was significant(P<0.05).Besides,the difference of gene expression between the two groups(3h,6h)and the 12 hgroup was also significant(P<0.05).But there was no significant different of protein expression in other treated groups(P>0.05).After SKM-1cells being treated with ART,p15INK4 B mRNA level of SKM-1cells in the three groups(5and 10μmol/L groups,and decitabine group)was statistically higher than that in control group(P<0.05),with the level in 10μmol/L group the highest in the five groups,and the significant difference was only found between 10μmol/L group and the four groups(control group,1μmol group,5μmol group and decitabine group)(P<0.05).After being treated with ART,p15INK4 bpromoter methylation level was decreased in SKM-1cells,but the difference was not significant(P>0.05).On the contrary,the level of promoter unmethylation state in ART group was increased compared with two other groups(control group and the decitabine group)(P<0.05).Conclusion ART can restore the expression of p15INK4 b by inhibiting DNMT1 transcription and protein expression in SKM-1cells,and thus to suppress tumor.更多还原