CAJ | 학술논문

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.
목적 탐토갑기전이매억제제5-담-2-탈양포감(DAC혹5-aza-dc)대담관암세포QBC939생장적영향급기궤제.방법 응용새서람(MTT)비색법검측DAC재불동농도(0.1、0.5、1.0、5.0、25.0、100.0μmol/L)급불동시간(24、48、72 h)하대담관암세포QBC939존활솔적영향,투사전경관찰세포형태학개변,류식세포의관찰세포생장주기급조망솔적변화;갑기화취합매련반응검측DAC작용전후p53-bax조망통로DNA갑기화변화.결과 DAC대QBC939세포생장적억제유농도화시간의뢰성,기반수억제솔(IC50)위72h 5μmol/L;경DAC작용후전경하담관암세포조망증가;담관암세포조망발생솔유DAC처리전적4.31%상승지처리후적43.04%;DAC작용전p53-bax조망통로중p14、DAPK억암기인갑기화,DAC작용후p14、DAPK억암기인탈갑기화.결론 DAC대담관암QBC939세포적생장구유억제작용,저일억제작용시유우DAC탈갑기화조성적,DAC대담관암QBC939세포p53-bax선립체조망통로DNA갑기화적영향사세포조망공능득이회복.
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.