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目的 :探讨HL 6 0细胞降钙素基因高甲基化的发生机制。方法 :采用HL 6 0细胞 ,以正常骨髓单个核细胞为对照 ,应用甲基化敏感的限制性内切酶消化 ,结合设有内外参照的PCR技术 ,检测CT基因甲基化状态 ;应用同位素微量分析法检测DNA 胞嘧啶 甲基转移酶 (DNA cytosin methyltransferase,MTase)活性 ;应用RT PCR技术检测MTase基因表达水平。结果 :HL 6 0细胞经甲基化敏感的内切酶HpaⅡ消化后仍可扩增出清晰的CT基因特异条带 ,提示CT基因 5′端呈高甲基化状态 ;HL 6 0细胞MTase活性平均测定值为 5 39.9Bq ,是正常骨髓单个核细胞 (平均 2 1 8Bq)的 2 9.8倍 ;MTase基因的mRNA表达水平以MTase与β actin内参照RT PCR产物的比值表示 ,HL 6 0细胞 (0 .72 )为正常对照 (0 .0 2 )的 36倍。结论 :HL 6 0细胞CT基因呈高甲基化状态 ,MTase活性增高可能是其发生机制之一
Objective: To investigate the mechanism of hypermethylation of calcitonin gene in HL-60 cells. Methods: The normal bone marrow mononuclear cells (HL 6 0) cells were used as control. The methylation status of CT gene was detected by methylation-sensitive restriction endonuclease digestion and combined with internal and external reference PCR. The activity of DNA cytosine methyltransferase (MTase) was detected by the method of analysis. The expression of MTase gene was detected by RT-PCR. Results: The clear bands of CT gene could still be amplified after digested with methylation-sensitive endonuclease HpaⅡ, indicating that the 5 ’end of CT gene was hypermethylated, and the average MTase activity of HL 60 cells was determined Value of 39.9Bq, which is 9.8 times higher than that of normal bone marrow mononuclear cells (average 2 1 8Bq). The MTase gene mRNA expression level is expressed as the ratio of MTase to β-actin RT PCR product, 72) was 36 times of normal control (0.02). Conclusion: The CT gene of HL 60 cells is hypermethylated and the increase of MTase activity may be one of the mechanisms