摘要
本研究通过对急性白血病病人及体外肿瘤细胞株的DNA甲基化及基因表达的实验,探讨死亡相关蛋白激酶(death-associated protein kinase,DAPK)基因在急性白血病中的表达情况及其启动子区域甲基化的状态,并分析两者之间的相关程度。应用RT-PCR检测DAPK基因在白血病细胞及正常骨髓细胞中的表达;用巢式甲基化特异性PCR(n-MSP)法检测急性白血病患者及细胞株DAPK基因启动子甲基化状况;从第二轮巢式MSP扩增出的随机产物中挑选2个随机引物,交予专业机构克隆后进行序列分析。结果表明:DAPK基因在10例正常对照者骨髓标本中均有表达,DAPK mRNA在急性白血病病人骨髓标本中表达平均值为0.61±0.40,低于正常对照者,差异有显著性(P<0.05),其中52.94%(9/17例)的急性淋巴细胞白血病病人骨髓标本DAPK mRNA表达降低或缺失,急性髓系白血病病人数据为41.18%(42/102);细胞株U-937显示基因表达正常,HL-60表达缺失;102例急性髓系白血病病人骨髓标本中33例存在DAPK启动子区甲基化,甲基化率为32.4%(33/102);17例急性淋巴细胞白血病病人骨髓标本中8例存在DAPK启动子区甲基化,甲基化率为47%(8/17);7例正常人骨髓标本DAPK启动子区均为非甲基化;细胞株U-937 DAPK启动子区非甲基化,HL-60 DAPK启动子区甲基化。ALL组与AML组患者骨髓细胞DAPK mRNA表达与其启动子甲基化均呈显著负相关(ALL组r=-0.855,P<0.05;AML组r=-0.343,P<0.05),提示两者有密切的关联。结论:急性白血病患者中DAPK基因启动区甲基化与其mRNA的异常表达或失表达有关。
This study was purposed to investigate the expression of death-associated protein kinase （DAPK） gene in acute leukemia （AL） patients and the methylation status of its promoter region through experiments of DAPK methylation and expression,and to analyze the relation between them.The expression of DAPK gene in leukemia ceils and normal bone marrow cells was detected by RT-PCR; the methylation status of DAPK gene promoter region in cells from AL patients and leukemia cell lines HL-60 and U937 was detected by nested methylation specific PCR （n-MSP） ; 2 randomy primers selected from randomy amplified products of second round nMS-PCR were cloned and sequenced in professional company.The results showed that the DAPK gene expressed in bone marrow specimens of all 10 normal controls,with average value of expression 0.92 ± 0.18,while the average value of DAPK expression in bone marrow specimens of AL patients was 0.61 ± 0.40 which was lower than that in normal controls （P ＜ 0.05）.The low or deletion of DAPK mRNA expression were found in bone marrow specimens of 9/17 （52.94％） cases of ALL and 42/ 102 （41.18％） cases of AML.The cell line U937 showed normal expression of DAPK gene,while cell line HL-60 showed the expression detetion of DAPK gene.The methylation of DAPK promoter region existed in 33 out of bone marrow specimens of 102 AML patients and in 8 out of bone marrow specimens of 17 ALL patients,the methyletion rates were 32.4％ （33/102） and 47％ respectively.The DAPK promoter region in bone marrow of 7 normal controls was unme-thylated,while DAPK promoter region in U937 cells and HL-60 cells were unmethylated and methylated respectively.The DAPK mRNA expression in ALL and AML patients significantly negatively correlated with the methylation of its promoter region （r =-0.855,P ＜0.05,in AML patients and r =-0.343,P ＜0.05,in AML patients）suggeting the close relationship between them.It is concluded that the methylation of DAPK gene promoter region relates with abnormal expression or detetion of DAPK mRNA in AL patients.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2014年第1期30-34,共5页
Journal of Experimental Hematology
