氯乙烯对大鼠肝细胞RASSF1A基因DNA甲基化的影响  

DNA methylation of RASSF1A induced by VC in rats

作　　者：续志斌 梁洁[1] 张林[1] 马文彦[1] 郑金平[1] 张文平[1] 阎小艳 仇玉兰[1] 

机构地区：[1]山西医科大学公共卫生学院卫生毒理教研室,山西太原030001

出　　处：《毒理学杂志》2017年第1期20-24,共5页Journal of Toxicology

基　　金：山西省基础研究计划项目(2013011059-1);山西省回国留学人员科研资助项目(2016-056)

摘　　要：目的通过检测氯乙烯(VC)染毒大鼠肝细胞RASSF1A基因启动子区甲基化的水平及mRNA的表达量,探讨RASSF1A基因在氯乙烯致癌过程中的表观遗传机制。方法选取96只健康雄性SD大鼠,随机分为4组:阴性对照组(0mg/kg)和低剂量(5 mg/kg)、中剂量(25 mg/kg)、高剂量(125 mg/kg)3个氯乙烯染毒组。腹腔注射,隔日染毒,每周3次。每组分别于6、8和12周随机处死8只,取其肝脏。采用甲基化特异性实时荧光定量(QMSP)检测大鼠肝细胞RASSF1A基因启动子区甲基化的水平,采取实时荧光定量(QPCR)测定RASSF1A mRNA表达量的改变。结果染毒6周时,RASSF1A基因启动子区甲基化水平各组间无明显变化,差异无统计学意义(P>0.05);但RASSF1A mRNA的表达量随着剂量的增加而升高,且高剂量组、中剂量组与对照组相比,差异有统计学意义(P<0.05)。染毒8周时,RASSF1A基因启动子区甲基化水平和mRNA的表达量在各组间差异无统计学意义(P>0.05)。染毒12周时,RASSF1A基因启动子区甲基化水平随着染毒剂量的增加而明显升高,高剂量组与其他各组相比,差异有统计学意义(P<0.05);mRNA的表达量随着染毒剂量的增加而出现下降,且对照组和低剂量组与高剂量组相比,差异有统计学意义(P<0.05)。此外,在高剂量组中,8周时RASSF1A启动子区甲基化水平较6周时低,差异有统计学意义(P<0.05),而12周时启动子区甲基化水平较6周时高,且差异有统计学意义(P<0.05);mRNA表达量随着染毒时间的延长而下降,且8和12周mRNA表达量与6周时相比,差异有统计学意义(P<0.05),在其余各组中,不同染毒时间下RASSF1A甲基化水平及mRNA表达量均未出现统计学差异(P>0.05)。结论氯乙烯长期染毒可致大鼠肝细胞RASSF1A基因启动子区甲基化水平的升高,并引起其mRNA的表达量下降,可能参与氯乙烯致癌的表观遗传机制。Objective Detecting aberrant methylation in the promoter and mRNA expression of RASSF1A in rat liver to discuss the epigenetic mechanism of carcinogenic process of vinyl chloride. Method Ninety-six healthy SD male rats were randomly divided into a negative control group（ 0 mg/kg） and three VC exposed groups（ 5 mg/kg,25 mg/kg,125 mg/kg）. Different doses of VC were given to SD rats by intraperitoneal injection every other day,totally three times a week. Eight rats in each group were randomly killed at the end of 6,8and 12 weeks respectively and the livers were taken. The methylation status of the 5＇region of RASSF1A were detected by Quantitative Realtime methylation specific PCR（ QMSP）. The mRNA expressions of RASSF1A were examined by Real-time PCR（ QPCR）. Result There was no significant differnce of hypermethylation and mRNA expression level in all groups at the end of 6th and 8th week（ P〉0. 05）. But the RASSF1A mRNA expression level and methylation were increased as dose increased,and there were significant difference in middle and high dose group compared with the control group in 6th week（ P〈0. 05）. The characteristics mentioned above had no difference among the groups in 8th week（ P〉0. 05）. The RASSF1A mRNA methylation level in 12th week was also increased as dose increased,there was significant difference in high dose group compared with the others. And the mRNA expression decreased as the dose increased,and the difference was statistically significant. The RASSF1A mRNA methylation level in 6th week was higher than that in 8th week,but lower than that in 12th week（ P〈0. 05）. The mRNA expression level decreased with the prolonged exposure time,the difference was statistically significant in 8th and 12th week,compared with the 6th week. And there were no significant difference of RASSF1A mRNA methylation and expression level in other groups（ P〈0. 05）. Conclusion These findings suggest hypermethylation of 5＇region of RASSF1A can be induced by VC. And the hypermet

关 键 词：氯乙烯(VC) RASSF1A DNA甲基化 mRNA表达 

分 类 号：R114[医药卫生—卫生毒理学]