孕期酒精暴露致DNA甲基化异常对子代小鼠心脏发育相关基因表达的影响  

Effects of Alcohol Consumption During Pregnancy on DNA Methylation and the Cardiomyogenesis Related Gene Expression in the Heart of Offspring Mice

作　　者：罗孝美 韩笑 彭昌 邓玲 黄丽欣 LUO Xiaomei;HAN Xiao;PENG Chang;DENG Ling;HUANG Lixin(Department of Physiology,School of Basic Medical Sciences,Zunyi Medical University,Zunyi(563000),Guizhou,China)

机构地区：[1]遵义医科大学基础医学院生理学教研室,贵州省遵义市563000 [2]遵义医科大学附属医院小儿内科

出　　处：《中国循环杂志》2020年第3期299-304,共6页Chinese Circulation Journal

基　　金：贵州省科技计划项目[黔科合基础(2016)1177];遵义医科大学博士启动基金项目[院字(2015)4号]

摘　　要：目的:探讨DNA甲基化修饰失衡在孕期酒精暴露致子代小鼠心脏发育相关基因表达异常中的作用,为防治孕期饮酒所致的心脏发育畸形提供新思路。方法:选取24只健康昆明孕鼠按照随机数字表法等分为四组:正常组、对照组、酒精组、干预组。从孕期0.5 d^16.5 d,酒精组每日给予56%酒精(5 ml/kg)灌胃1次,干预组在上述酒精灌胃基础上给予每日1次腹腔注射DNA甲基转移酶(DNMT)抑制剂5-氮杂胞苷(2.5 mg/kg),对照组给予等量生理盐水灌胃和等量二甲基亚砜(DMSO)腹腔注射,正常组未予任何处理。于胎龄16.5 d收集各组子代小鼠心脏进行以下检测:(1)比色法检测DNMT活性;(2)甲基化测序检测心脏核心转录因子心肌细胞增强因子2A(MEF2A)启动子区CpG岛DNA甲基化水平,实时荧光定量PCR检测MEF2A转录水平;(3)染色质免疫共沉淀(ChIP)检测MEF2A对心脏结构基因心房利钠肽(ANP)、β肌球蛋白重链(β-MHC)和心肌肌钙蛋白T(cTnT)的调控作用;(4)蛋白免疫印迹法(Western blot)检测心脏结构基因ANP、β-MHC及cTnT的蛋白表达水平。结果:(1)比色法结果显示,酒精组及干预组胎鼠心肌组织中DNMT活性较对照组和正常组显著降低,差异有统计学意义(P<0.05);(2)心脏核心转录因子MEF2A启动子区CpG岛DNA甲基化水平在酒精组及干预组较对照组和正常组均显著降低(P<0.05);(3)实时荧光定量PCR结果显示,心脏核心转录因子MEF2A转录水平在酒精组及干预组较对照组和正常组显著升高(P<0.05);(4)ChIP结果表明,心脏核心转录因子MEF2A可结合心脏结构基因ANP、β-MHC及cTnT启动子区域直接参与上述基因的表达调控;(5)Western blot结果表明,酒精组和干预组小鼠心肌组织中ANP、β-MHC及cTnT的蛋白表达水平较对照组和正常组显著升高,差异有统计学意义(P均<0.05)。结论:酒精介导的DNA低甲基化可能参与了孕期酒精暴露所致的子代心脏发育相关基因表达异常。Objectives:To investigate the changes of DNA methylation and cardiomyogenesis related gene expression in the heart of the offspring mice induced by alcohol consumption during pregnancy,and to provide experimental evidence for potential options on preventing and treating cardiac development malformation in offspring induced by alcohol consumption during pregnancy.Methods:Pregnant Kunming mice were divided into four groups(n=6 each)according to random number table method:normal group,control group,alcohol group,intervention group.56%alcohol(5 ml/[kg·d])was given by gavage in mice assigned for alcohol group during embryo(E)0.5-16.5 days;5-azacytidine(2.5 mg/kg),a DNA methyltransferases(DNMTs)inhibitor,was intraperitoneally injected into pregnant mice on top of alcohol for up to E16.5,and the control group was treated with equal volume saline and dimethylsulfoxide(DMSO),and the normal group did not receive any treatment.The hearts of fetal mice in E 16.5 were collected for analysis.The DNMTs activity were tested by colorimetry,and the level of methylation in the CpG island of MEF2A promoter were assayed using next generation sequencing,and transcription level of MEF2A were tested by real-time PCR.The regulatory relationship between heart nucleus transcription factor MEF2A and heart development related structural genes(ANP,β-MHC and cTnT)were tested by ChIP.The protein expression of ANP,β-MHC and cTnT were assayed by Western blot in the heart of fetal mice.Results:The data of colorimetry showed that the activity of DNMTs was significantly lower in alcohol group and intervention group than in control group and normal group(P<0.05).The level of DNA methylation in the CpG island of MEF2A promoter was lower in alcohol group and intervention group than in control group and normal group(P<0.05).The transcription level of MEF2A was higher in alcohol group and intervention group than in control group and normal group(P<0.05).ChIP data showed that heart nucleus transcription factor MEF2A could directly regulate heart struct

关 键 词：DNA甲基化 心脏核心转录因子 胎鼠 酒精 

分 类 号：R541[医药卫生—心血管疾病]