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ApoE-/-小鼠(ApoE-/-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 mRNA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(pEGFP-N1组)和重组质粒组(pEGFP-N1-DNMT1组),NC组用不做任何处理,继续培养,pEGFP-N1组加入10μl脂质体Lipofectamine和20μg空质粒载体pEGFP-N1,pEGFP-N1-DNMT1组加入10μl脂质体Lipofectamine和20μg pEGFP-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,ApoE-/-组TC、LDL、TG呈升高趋势,HDL无明显变化;各时段ApoE-/-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,ApoE-/-组主动脉内膜明显增厚并伴炎症浸润.第8、12、16及20周,ApoE-/-组MDA、ROS呈升高趋势,各时段ApoE-/-组MDA、ROS高于WT组.ApoE-/-组EC-SOD mRNA表达水平、DNMT1 mRNA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 mRNA表达水平低于WT组,DNMT1 mRNA表达水平和EC-SOD甲基化水平高于WT组.pEGFP-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pEGFP-N1组和NC组比较,差异有统计学意义(P <0.05),pEGFP-N1-DNMT1组DNMT1蛋白表达、EC-SOD甲基化水平高于pEGFP-N1组和NC组,EC-SOD蛋白表达水平低于pEGFP-N1组和NC组,pEGFP-N1组和NC组EC-SOD、DNMT1蛋白及EC-SOD甲基化水平比较差异无统计学意义(P >0.05).结论 EC-SOD甲基化程度升高,导致EC-SOD表达降低,机体抵抗氧化应激反应能力减弱,最终发展成动脉粥样硬化.%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 10 μl liposome Lipofectamine and 20 μg of empty plasmid vector pEGFP-N1), pEGFP-N1-DNMT1 group (added with 10 μl liposome Lipofectamine and 20 μg recombinant plasmid vector pEGFP-N1-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 significantly higher than those of the pEGFP-N1 group and the NC group, EC-SOD protein level was significantly lower than that of the pEGFP-N1 group and the NC group (P < 0.05). EC-SOD and DNMT1 protein levels and EC-SOD methylation level had no statistically differences between the pEGFP-N1 group and the NC group (P > 0.05). Conclusions Increase of EC-SOD methylation can lead to decrease of EC-SOD expression and weakening of body resistance to oxidative stress reaction, and ultimately results in development of atherosclerosis.
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