【摘要】 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更多还原

【Abstract】 Objective: To explore the mechanism of hypermethylation pattern of the calcitonin (CT) gene in the HL 60 cells. Methods: Methylation pattern of CT gene in the HL60 cells was detected by PCR technique combined with methylation sensitive restriction endonuclease digestion. DNA Cytosine Methyltransferase (MTase) activity and mRNA expression of MTase gene were detected by isotope labeled microassay and RT PCR, respectively. Results: A specific band of CT gene was obtained after Hpa Ⅱ digestion, which suggested hypermethylation in 5′ region of CT gene in HL 60 cells. The MTase activity in the HL 60(mean 539.9 Bq) was 28.8 fold higher than that of the control (mean 21.8 Bq).The level of MTase gene expression was evaluated by the ratio of density between MTase band and β actin band. The ratio of the HL 60 cells (0.72) was 36 fold higher than that of the control (0.02). Conclusion: Increased MTase activity might be one of possible mechanisms of CT gene hypermethylation in the HL 60 cells.更多还原