5-Aza-CdR对膀胱癌EJ细胞系生长及Apaf-1基因异常甲基化的影响

目的观察5-氮杂-2′-脱氧胞苷（5-aza-2-′deoxyc ityd ine，5-Aza-CdR）对体外培养的膀胱癌EJ细胞增生、细胞周期影响及其对此细胞株中Apaf-1基因的甲基化状态的影响。方法不同浓度5-Aza-CdR处理体外培养膀胱癌EJ细胞后，用MTT法检测药物处理24、48、72 h后的细胞增殖活性；PI染色和流式细胞仪检测药物处理72 h后的细胞周期分布；MSP法检测用药前后细胞中Apaf-1基因的甲基化状态；Real-time RT-PCR法检测用药前后细胞中Apaf-1的mRNA表达变化。结果2×10^-6、5×10^-6、1×10^-5mol/L 5-Aza-CdR处理膀胱癌EJ细胞24、48、72 h后，细胞增生受到抑制，有时间和剂量依赖性。流式细胞仪分析表明，不同药物浓度处理EJ细胞72 h后细胞增殖指数降低明显：5×10^-6、1×10^-5mol/L组分别为（34.09±0.79）、（28.55±1.76），与对照组（42.78±1.23）相比，差异有统计学意义（P〈0.05）。在5-Aza-CdR处理前未检测到膀胱癌EJ细胞系的Apaf-1基因的mRNA表达，经过5-Aza-CdR处理后，Apaf-1基因在膀胱癌EJ细胞系中甲基化状态得到了逆转，Apaf-1基因的mRNA重新表达。结论5-Aza-CdR对膀胱癌EJ细胞具有增生抑制作用；Apaf-1基因的表达情况与其甲基化状态的改变有关。

Effects of 5-Aza-CdR on proliferation of EJ human bladder cancer cell line and abnormal methylation of Apaf-1 gene

Objective To observe the effects of 5-aza-2＇-deoxycitydine （5-Aza-CdR） on the proliferation of EJ human bladder cancer cell line as well as on the methylation and expression of Apaf-1 gene. Methods Human bladder cancer cell line were cultured in RPMI1640 and then treated with different concentrations of 5-Aza-CdR. The proliferation of the cells was detected by MTT assay and flow cytometry （FCM） after being cultured for 24, 48 and 72 h. The methylation of Apaf-1 gene in the cell line was detected by methylation- specific-polymerase chain reaction （ MSP）, and the expression of Apaf-1 was detected by real-time RT-PCR. Results 5-Aza-CdR displayed a growth inhibitory effect on EJ cell in a dose-and time-dependent manner after exposure to 5-Aza-CdR at different concentrations （2 ×10^-6,5 × 10^-6, and 1 × 10^-5 mol/L） for 24, 48 and 72 hours. FCM analysis showed that the proliferation index （PI） in EJ cells （34.09 ± 0.79, 28.55 ±1.76 ） decreased significantly after cell＇ s exposure to 5-Aza-CdR （5 ×10^-6, 1 ×10^-5 moL/L） for 72 hours as compared with that （42.78 ± 1.23 ） in control cells （ P 〈 0.05 ）. The methylation and loss of Apaf-1 mRNA expression were detected in EJ cells before 5-Aza-CdR treatment. However, the methylation was reversed and Apaf-1 was re-expressed after 5-Aza-CdR treatment. Conclusion 5-Aza-CdR can inhibit the proliferation of EJ cells through blocking cell cycles and inducing cell apoptosis, during which the reversion of Apaf-1 gene methylation plays an important role.