摘要目的 探讨三氧化二砷(As2O3)诱导人视网膜母细胞瘤(RB)Y79细胞系P16基因去甲基化及转录激活的可能机制. 方法四甲基偶氮唑盐微量酶反应比色法(MTT)法检测As2O3对Y79细胞生长和增生的抑制作用;流式细胞仪DNA含量分析法探讨As2O33对Y79细胞周期的影响;巢式甲基特异性聚合酶链反应法(n-MSP)及DNA克隆测序分析法检测As2O3作用前后Y79细胞P16基因甲基化状态;逆转录聚合酶链式反应(RT-PCR)检测P16基因、DNA甲基转移酶(DNMT3A、DNMT3B)基因mRNA的表达. 结果 As2O3能明显抑制Y79细胞生长,使细胞阻滞于G0～G<,1>期;未处理组细胞P16基因不表达,As2O3作用48 h后P16基因表达增强,甲基化程度减弱,甲基转移酶DNMT3A、DNMT3B mRNA表达下降. 结论 RB Y79细胞系存在P16基因高甲基化,As2O3通过抑制DNA甲基转移酶mRNA表达诱导P16基因去甲基化,显著上调P16基因mRNA表达,将细胞阻滞于G0～G<,1>期,抑制Y79细胞增生.

abstractsObjective To investigate the possible mechanism of arsenic trioxide (As2O3) inducing P16 gene demethylation and transcription regulation in the retinoblastoma (RB) Cell Line Y79. Methods The induced growth inhibition of Y79 cell was assayed by MTT;The DNA content of Y79 cell was analyzed by flow cytometry after being exposed to As2O3;the methylation status of the P16 gene in Y79 cell line before and after treatment with As2O3 was detected by the nested-methylation specific PeR and DNA sequencing;the mRNA of P16, DNA methyltransferases (DNMT3A and 3B)gene were determined by RT-PCR. Results As2O3 was able to inhibit the growth of Y79 cell and increase the cell number in G0-G1phase;P16 gene was not expressed in Y79 cell line and As2O3 can induce it's mRNA expression;after 48-hour disposal of As2O3,the methylation levelof P16 gene was apparently attenuated in Y79 cell line, the expression of DNMT3A and DNMT3B was obviously down-regulated. Conclusions P16 gene is the hypermethylation in the retinoblastoma cell line Y79, and As2O3 can inhibite the methylation of P16 gene and up-regulate the expression of p16 gene mRNA which inhibits the proliferation of Y79 cell by inducing the G0-G1arrest, by inhibiting the expression of DNA methyltransferases.