二乙基亚硝胺诱导大鼠肝癌组织CLDN1基因表达及其甲基化  

Detection of the expression and promoter methylation of CLDN1 in rat models of hepatocellular carcinoma induced by diethylnitrosamine

作　　者：张斌[1] 李琦[2] 殷佩浩[2] 高虹[2] 赵成根[2] 范忠泽[2] 

机构地区：[1]复旦大学附属中山医院青浦分院肝病科,上海201700 [2]上海中医药大学附属普陀医院中医肿瘤科,上海200062

出　　处：《中国实验动物学报》2013年第1期80-83,F0003,共5页Acta Laboratorium Animalis Scientia Sinica

基　　金：上海市普陀区科委自主创新基金课题资助项目(普科委200937);上海市嘉定区卫生局基金资助(NO.KYXM2010-12-03)

摘　　要：目的探讨二乙基亚硝胺(diethylnitrosamine,DEN)诱导大鼠肝癌发生中肝癌组织CLDN1基因表达及其启动子甲基化的规律。方法 65只雄性Wistar大鼠随机选择40只作为模型组,其余作为正常组。模型组在1～12周饮用含DEN 80 mg/L的饮水以诱癌(每日8mg/kg),各组在造模过程的第4周、8周、12周、16周随机5只取肝,第20周剩余大鼠取肝,应用RT-PCR方法检测肝组织CLDN 1 mRNA的表达,应用MSP法检测肝组织CLDN1启动子甲基化和非甲基化。结果模型大鼠病死率为10%(4/40),正常组无死亡。至第20周,成瘤率达到100%。RT-PCR显示,与正常组比较,模型组在16周和20周CLDN1mRNA表达下调(P<0.05),其他各周两组差异不显著。MSP结果表明,模型组肝组织CLDN1甲基化率达77.78%,而正常肝组织甲基化率为24%,两者比较差异有显著性(P<0.01)。结论 CLDN1启动子甲基化及CLDN1基因表达下调与大鼠肝癌病变相关,对其机制值得进一步深入研究。Objective To investigate the expression mechanism of CLDN1 and its promoter methylation status in rat models of hepatoccllular carcinoma induced by diethylnitrosamine. Methods Male Wistar rats were randomly divided into normal control group （n =25） and model group （n =40）. The rats of model group were given DEN drinking water （80 ppm） at a dose of 8 mg/（kg.d） for continuous 12 weeks to induce hepatoma. Each group was randomly executed 5 rats at the 4th, 8th, 12th, 16th week, respectively, and other rats were all killed at the 20th week. The expression of CLDN1 mRNA was determined by RT-PCR. At the same time, the promoter methylation of CLDN1 was tested by MSP methods. Results At the end of 20th week, the mortality rate of normal control group was 0 and that of the model group was 10. 0% , and the tumor formation rate of model group at 20th week was 100%. RT-PCR analysis showed that the CLDN1 mRNA expression of the model group was declined only at 16th week and 20th week （P 〈 0. 05 vs. normal group）. MSP analysis showed that the CLDN1 methylation rate of the model group was 77.78% ,while that of the normal group was only 24% （P 〈0.01 ）. Conclusions The decreased expression of CLDN1 and high methylation occurrence in the promoter region of CLDN1 are closely related with the pathogenesis of liver cancer induced by diethylnitrosamine, and its mechanism is worth further studying.

关 键 词：原发性肝癌 二乙基亚硝胺 大鼠 CLDN1 DNA甲基化 

分 类 号：Q95-33[生物学—动物学] R730.2[医药卫生—肿瘤]