细胞外超氧化物歧化酶DNA甲基化对动脉粥样硬化的诱导作用及作用靶点  

Role of extracellular superoxide dismutase methylation in atherosclerosis

作　　者：陈佳[1] 杨晓龙[2] 张旭峰[3] Jia Chen;Xiao-long Yang;Xun-feng Zhang(Emergency Department,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;ICU,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Clinical Laboratory,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院急诊科,上海201203 [2]上海中医药大学附属曙光医院重症医学科,上海201203 [3]上海中医药大学附属曙光医院检验科,上海201203

出　　处：《中国现代医学杂志》2018年第10期18-25,共8页China Journal of Modern Medicine

摘　　要：目的探讨细胞外超氧化物歧化酶DNA(EC-DNA)甲基化水平改变在动脉粥样硬化发病中的作用。方法 16只Apo E-/-小鼠(Apo E-/-组)给予高脂饲料喂养,16只C57BL/6小鼠(WT组)给予不含任何高脂成分的普通饲料喂养,分别于喂养第8、12、16和20周检测小鼠血脂、主动脉组织形态及丙二醛(MDA)、活性氧(ROS)含量,采用逆转录聚合酶链反应(RT-PCR)检测主动脉组织细胞外超氧化物歧化酶(EC-SOD)、DNMT1 m RNA水平,巢式甲基化特异性聚合酶链反应检测主动脉组织EC-SOD DNA甲基化程度。将THP-1诱导为巨噬细胞,分为3组:空白对照组(NC组)、空白质粒组(p EGFP-N1组)和重组质粒组(p EGFP-N1-DNMT1组),NC组用不做任何处理,继续培养,p EGFP-N1组加入10μl脂质体Lipofectamine和20μg空质粒载体p EGFP-N1,p EGFP-N1-DNMT1组加入10μl脂质体Lipofectamine和20μg p EGFP-N1-DNMT1重组质粒载体,培养24 h后检测细胞EC-SOD、DNMT1蛋白表达和EC-SOD DNA甲基化程度。结果第8、12、16和20周,Apo E-/-组TC、LDL、TG呈升高趋势,HDL无明显变化;各时段Apo E-/-组TC、LDL、TG、HDL与WT组比较,差异有统计学意义(P<0.05),TC、LDL、TG高于WT组,HDL低于WT组。第16和20周,WT组动脉组织内皮细胞结构规则有序,Apo E-/-组主动脉内膜明显增厚并伴炎症浸润。第8、12、16及20周,Apo E-/-组MDA、ROS呈升高趋势,各时段Apo E-/-组MDA、ROS高于WT组。Apo E-/-组EC-SOD m RNA表达水平、DNMT1 m RNA表达水平和EC-SOD甲基化水平与WT组比较,差异有统计学意义(P<0.05),EC-SOD m RNA表达水平低于WT组,DNMT1 m RNA表达水平和EC-SOD甲基化水平高于WT组。p EGFP-N1-DNMT1组DNMT1蛋白表达、EC-SOD甲基化水平及EC-SOD蛋白表达水平与p EGFP-N1组和NC组比较,差异有统计学意义(P<0.05),p EGFP-N1-DNMT1组DNMT1蛋白表达、EC-SOD甲基化水平高于p EGFP-N1组和NC组,EC-SOD蛋白表达水平低于p EGFP-N1组和NC组,p EGFP-N1组和NC组EC-SOD、DNMT1蛋白及EC-SOD甲基化水平比较差�Objective To explore the mechanism of extracellular superoxide dismutase(EC-SOD)DNA methylation changes in atherosclerosis.Methods Sixteen ApoE-/-mice(ApoE-/-group)were fed with high-fat diet,and 16 C57BL/6 mice(WT group)were fed with ordinary diet without any high-fat component.The serum lipid level,aortic morphology and tissue MDA and ROS content were detected in the 8th,12th,16th and 20th weeks after feeding respectively.The EC-SOD and DNA methyltransferase 1(DNMT1)mRNA levels in the aortic tissues were detected by RT-PCR.EC-SOD DNA methylation level was detected by nested methylation specific PCR.THP-1 was induced into macrophages and divided into three groups:NC group(without any treatment,continue to cultivate),pEGFP-N1 group(added with 10μl liposome Lipofectamine and 20μg of empty plasmid vector pEGFP-N1),pEGFP-N1-DNMT1 group(added with 10μl liposome Lipofectamine and 20μg recombinant plasmid vector pEGFPN1-DNMT1),The EC-SOD and DNMT1 protein expressions and EC-SOD DNA methylation level were detected after culture for 24 h.Results In the 8th,12th,16th and 20th weeks,TC,LDL and TG of the ApoE-/-group were in increasing trends,and HDL had no significant change,the TC,LDL and TG of the ApoE-/-group were significantly higher than those of the WT group,HDL was significantly lower than that of the WT group(P<0.05).In the 16th and 20th weeks,the endothelial cells of artery tissue were regularly and orderly arranged in the WT group,while in the ApoE-/-group aortic intima was obviously thickened with inflammatory infiltration.In the 8th,12th,16th and 20th weeks,MDA and ROS of the ApoE-/-group were in increasing trends,the MDA and ROS content of the ApoE-/-group was significantly higher than that of the WT group(P<0.05).EC-SOD mRNA expression level of the ApoE-/-group was significantly lower than that of the WT group,DNMT1 mRNA level and EC-SOD methylation level were significantly higher than those of the WT group(P<0.05).DNMT1 protein expression and EC-SOD methylation level of the pEGFP-N1-DNMT1 group were sign

关 键 词：细胞外超氧化物歧化酶 DNA甲基化 DNA甲基转移酶 动脉粥样硬化 氧化应激损伤 

分 类 号：R-332[医药卫生] R363