5-氮-2-脱氧胞苷调控p53-bax凋亡通路DNA甲基化对胆管癌细胞生长的影响  

Effects of methylation of p53-bax mitochondrial apoptosis pathway regulated by 5-aza-2-deoxycyti-dine on the growth of cholangiocarcinoma cells

作　　者：刘小方[1] 姜宏[1] 张翠生[1] 于绍平[1] 王在秋[1] 苏海龙[1] 

机构地区：[1]青岛大学医学院附属烟台毓璜顶医院肝胆外科,烟台264000

出　　处：《中华实验外科杂志》2010年第7期876-878,共3页Chinese Journal of Experimental Surgery

基　　金：基金项目：山东省自然科学基金资助项目（Y2008C82）

摘　　要：目的 探讨甲基转移酶抑制剂5-氮-2-脱氧胞苷（DAC或5-aza-dc）对胆管癌细胞QBC939生长的影响及其机制.方法 应用噻唑蓝（MTT）比色法检测DAC在不同浓度（0.1、0.5、1.0、5.0、25.0、100.0μmol/L）及不同时间（24、48、72 h）下对胆管癌细胞QBC939存活率的影响,透射电镜观察细胞形态学改变,流式细胞仪观察细胞生长周期及凋亡率的变化；甲基化聚合酶链反应检测DAC作用前后p53-bax凋亡通路DNA甲基化变化.结果 DAC对QBC939细胞生长的抑制有浓度和时间依赖性,其半数抑制率（IC50）为72h 5μmol/L 经DAC作用后电镜下胆管癌细胞凋亡增加 胆管癌细胞凋亡发生率由DAC处理前的4.31%上升至处理后的43.04% DAC作用前p53-bax凋亡通路中p14、DAPK抑癌基因甲基化,DAC作用后p14、DAPK抑癌基因脱甲基化.结论 DAC对胆管癌QBC939细胞的生长具有抑制作用,这一抑制作用是由于DAC脱甲基化造成的,DAC对胆管癌QBC939细胞p53-bax线粒体凋亡通路DNA甲基化的影响使细胞凋亡功能得以恢复.Objective To study the effects of 5-aza-2-deoxycytidine （DAC or 5-aza-dc） , on the growth of cholangiocarcinoma cells （QBC939） and the mechansim. Methods Methyl thiazolyl tetrazolium （MTT） method was used to measure the growth of QBC939 cells treated with DAC at different concentrations （0.1, 0.5, 1.0, 5.0, 25. 0, 100.0μmol/L） and different time points （24, 48, 72 h）. The morphology was observed by electron microscopic technique （EM）. Cell growth cycle and apoptosis were examined by flow cytometry. Promoter hypermethylation of DAPK, pl4 and ASC genes were detected by methylation-specific polymerase chain reaction （PCR） before and after treatment with DAC. Results DAC inhibited QBC939 cells in concentration-and time-dependent manners, and its half inhibition ratio （IC50） was 5μmol/L at 72 h. After treatment with DAC, apoptosis was observed through scanning and transmission electronic microscope. Flow cytometric analysis demonstrated that the apoptosis rate of QBC939 cells was inhanced greatly from 4.31% to 43.04%. The tumor suppressor genes, pl4 and DAPK, in QBC939 cells were methylated before treatment with DAC, and demethylated after treatment with DAC. Conclusion DAC inhibits the growth of QBC939 cells because of demethylation. DAC affects methylation of p53-bax mitchondrial apoptosis pathway in cholangiocarcinoma cells and recovers cell apoptosis.

关 键 词：胆管癌 5-氮-2-脱氧胞苷 甲基化 脱噬作用 

分 类 号：R735.8[医药卫生—肿瘤]