As_2O_3对Jurkat细胞株hdpr1基因去甲基化作用机制的研究

本研究探讨三氧化二砷（arsenic trioxide,As2O3）对急性T淋巴细胞白血病Jurkat细胞株hdpr1抑癌基因去甲基化的影响及其作用机制。采用CCK8法检测As2O3对Jurkat细胞的增殖抑制作用,流式细胞术检测As2O3作用前后细胞周期的变化,甲基化特异性PCR（MSP）检测As2O3对hdpr1基因甲基化模式的影响,用半定量RT-PCR检测As2O3对hdpr1基因、DNA甲基转移酶基因dnmt1、dnmt3a、dnmt3b mRNA表达水平的影响。结果表明：As2O3可明显抑制Jurkat细胞的增殖并呈时间-浓度依赖性;As2O3阻滞Jurkat细胞周期于G0/G1期（p〈0.05）,呈浓度依赖性;As2O3能够逆转Jurkat细胞株hdpr1基因的高甲基化并诱导其mRNA重新表达,同时下调dnmt1、dnmt3a、dnmt3b mRNA的表达水平,亦呈浓度依赖性。结论：As2O3抑制Jurkat细胞的增殖,阻滞细胞周期于G0/G1期,其机制可能为下调dnmt1、dnmt3a、dnmt3b基因的表达,诱导Jurkat细胞中异常甲基化的hdpr1基因去甲基化并使其恢复表达。

Mechanism of As2O3 on hdpr1 Gene Demethylation in Jurkat Cell Line

This study was purposed to investigate the effect of As2O3 on the demethylation of anti-oncogene-hdpr1 gene of acute lymphoblastic leukemia cell line Jurkat in vitro and its mechanism.The inhibitory effect of As2O3 on the proliferation of Jurkat cells was assayed by CCK-8; the change of Jurkat cell cycle was detected by flow cytometry before and after using As2O3; the effect of As2O3 on the methylation model of hdpr1 gene was analyzed by methylation-specific PCR,and the effect of As2O3 on the expression of hdpr1 mRNA was analyzed by semiquantitative RT-PCR. The results showed that the proliferation rate of Jurkat cells was decreased significantly after being treated with As2O3,and in dose-and time-dependent manner; As2O3 blocked Jurkat cell cycle in G0/G1 phase in dose-dependent manner. As2O3 could reverse hypermethylation of hdpr1 gene and induce its mRNA reexpression,and down-regulate the dnmt1,dnmt3a,dnmt3b mRNA expression level also in dose-dependent manner. It is concluded that the As2O3 suppresses the proliferation of Jurkat cells and blocks cell cycle is G0/G1,its possible mechanism may be down-regulating mRNA expression level of dnmt1,dnmt3a and dnmt3b,induc demethylation of hdpr1 gene from abnormal hypermethylation status and activates its reexpression.