亚砷酸诱导人食管癌细胞株EC109细胞p15^(INK4B)基因的表达  

Expression of p15^(INK4B) gene induced by arsenic trioxide in esophageal cancer cell line EC109

作　　者：张学彦[1] 刘铁夫[2] 刘伟[3] 崔希威[4] 

机构地区：[1]哈尔滨医科大学附属二院消化科,黑龙江省哈尔滨市150086 [2]哈尔滨医科大学附属四院消化科,黑龙江省哈尔滨市150000 [3]哈尔滨医科大学附属二院分子实验中心,黑龙江省哈尔滨市150086 [4]哈尔滨医科大学附属二院计算机中心,黑龙江省哈尔滨市150086

出　　处：《世界华人消化杂志》2006年第19期1859-1863,共5页World Chinese Journal of Digestology

基　　金：黑龙江省科技计划攻关重点项目基金资助;No.GB05C401-09;黑龙江省卫生厅科研基金资助项目;No.2004-117

摘　　要：目的：研究亚砷酸(As_2O_3)对人食管癌EC109细胞株细胞周期蛋白依赖性激酶抑制因子p15^(INK4B)(p15)基因表达的影响．方法：终浓度2μmol/L的As_2O_3加入食管癌EC109细胞系,采用甲基化特异PCR(MSP)检测食管癌EC109细胞系中p15基因甲基化,采用RT-PCR和Western blot方法检测As_2O_3处理前后p15的mRNA和蛋白质水平的表达情况．用Scion Image软件测量条带灰度值,P15蛋白与ACTB条带的灰度比进行半定量分析．结果：EC109细胞p15基因发生高甲基化,p15基因不表达．As_2O_3作用后p15基因甲基化程度明显下降,As_2O_3作用72,48,24 h组和未加药组之间差异均有统计学意义(37．11±3．62,50．92±5．47,72．07±7．53 vs 97．23±9．80,P＜0．05)．As_2O_3作用24 h后出现p15 mRNA表达,随As_2O_3作用时间延p15 mRNA表达逐渐增强,各个时间组之间比较,除了未加药组和加药24 h组之间及加药24 h组和加药48 h组之间差异无统计学意义外,其余各个时间组之间差异有统计学意义(0．72±0．07 vs 0．58±0．06 vs 0．48±0．07 vs 0．41±0．08,P＜0．05)．As_2O_3作用24 h后P15蛋白出现表达,2μmol/L作用24-72 h中P15蛋白条带灰度逐渐增强,As_2O_3作用72,48,24 h组和未加药组之间差异均有统计学意义(0．51±0．02 vs 0．21±0．01 vs 0．16±0．02 vs 0．06±0．01,P＜0．05),说明其表达随As_2O_3作用时间延长而逐渐增强．结论：As_2O_3可使食管癌EC109细胞p15基因去甲基化,使p15基因表达上调,从而抑制细胞周期进程．AIM： To investigate the effects of arsenic trioxide （As2O3） on the expression of cyclin dependent kinase inhibitor p15^INK4B （p15） gene in esophageal cancer cell line EC109.METHODS： As2O3 was added to EC109 cells for succedent experiments （final concentration 2 μmol/L）. Methylation of p15 gene in EC109 cells was detected by polymerase chain reaction （PCR） using a methylation specific primer （MSP）, and the expression of p15 gene was detected by reverse transcription PCR （RT-PCR） and Western blot at the mRNA and protein level.RESULTS： p15 gene failed to express in EC109 cells due to hypermethylation. As2O3 （2 μmol/L） significantly down-regulate the methylation of p15 gene at DNA level, and the difference between As2O3 action 72, 48, 24 h and no drug groups had statistical significance （37.11± 3.62 vs 50.92 ± 5.47 vs 72.07 ± 7.53 vs 97.23 ± 9.80, P 〈 0.05）. After As2O3 action 24 h, p15 mRNA expression appeared. Along with the continuance of As2O3 action, the p15 mRNA expression became stronger. Except between no drug group and As2O3 action 24 h group as well as between As2O3 action 24 and 48 h group, the difference between all groups had statistical significance （0.72 ± 0.07, 0.58 ± 0.06, 0.48 ± 0.07 vs 0.41 ＋ 0.08, P 〈 0.05）. After As2O3 action 24 h, the expression of p15 protein appeared. Along with the continu- ance of As203 action, the p15 protein expression became stronger. The differences between all the groups were significant （0.51 ± 0.02 vs 0.21 ± 0.01 vs 0.16 ± 0.02 vs 0.06 ± 0.01, P 〈 0.05）.CONCLUSION： As2O3 can activate the expression of the p15 gene by demethylation and inhibit the cell cycle progress of esophageal cancer cell line.

关 键 词：亚砷酸 P15^INK4B 甲基化 食管癌 

分 类 号：R735.1[医药卫生—肿瘤]