目的 探讨肥胖儿童瘦素、瘦素受体及促阿片-黑素细胞皮质素原(proopiomelanocortin,POMC)基因启动子区CpG位点的甲基化改变。方法 选取北京酒仙桥地区45例4~6岁单纯性肥胖儿童及按性别、年龄1∶1 配对的45例正常儿童为研究对象。利用外周血提取基因组DNA,然后应用亚硫酸盐修饰直接测序法(BSP)和半巢式PCR检测儿童瘦素基因启动子区(324 bp,-228到+96)25个CpG位点、瘦素受体基因启动子区(271 bp,-411到-141)20个CpG位点及POMC基因启动子区(275 bp,-341到-67)18个CpG位点的甲基化状态;同时采用酶联免疫分析测定血浆瘦素含量。结果 单纯性肥胖儿童血浆瘦素含量(21.24±4.02) μg/L显著高于正常儿童(2.95±0.53) μg/L。肥胖儿童瘦素基因启动子区CpG位点的平均甲基化程度(0.43±0.13)显著低于正常儿童(0.52±0.15);对该启动子区单个CpG位点比较发现,9个位点的甲基化程度在肥胖组低于对照组。瘦素受体基因启动子区各CpG位点在两组儿童中均呈现完全去甲基化状态。POMC基因启动子区各CpG位点甲基化程度在肥胖与正常儿童之间均未见显著差异。结论 肥胖儿童瘦素基因启动子区CpG位点存在甲基化改变,可能与瘦素表达增加、瘦素抵抗发生有关。
Abstract
Objective To investigate the changes in promoter DNA methylation of satiety regulators,the leptin,leptin receptor (leptin-R) and proopiomelanocortin (POMC) in obese children. Methods 45 preschool obese children aged 4 to 6 years,and 45 age-and sex-matched normal-weight children were included in the study.Genomic DNA was extracted from peripheral white blood cells.Bisulfite sequencing-PCR was used to determine the CpG methylation of the leptin promoter (324 bp,-228 to +96),leptin-R promoter (271 bp,-411 to -141) and POMC promoter (275 bp,-341 to -67).Plasma leptin concentrations were measured by enzyme-linked immunosorbent assay. Results Plasma leptin concentration [(21.24±4.02) μg/L] in obese children was higher than that in normal children [(2.95±0.53) μg/L].The mean methylation fraction of 25 CpG sites in the leptin promoter in obese children (0.43±0.13) was lower than that in normal children (0.52±0.15),with significantly reduced methylation fraction in 9 CpG sites.For CpG sites in leptin-R promoter,the methylation fraction was completely demethylated in both obese and normal children.No differences were found in the methylation fraction of CpG sites between the two groups of children. Conclusion Reduced CpG methylation in the leptin promoter may associate with the increased leptin expression and resistance in obese children.
关键词
瘦素 /
瘦素受体 /
阿片-黑素细胞皮质素原 /
肥胖 /
DNA甲基化
Key words
leptin /
leptin receptor /
proopiomelanocortin /
obesity /
DNA methylation
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基金
北京市卫生系统高层次卫生技术人才培养计划(学科骨干)项目(2009-3-40)
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