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长链游离脂肪酸对滋养细胞线粒体三羟基酰基辅酶A脱氢酶基因启动子区甲基化程度的影响
其他题名Study on the methylation of LCHAD gene promoter region in mitochondria of trophoblast cells incubated with long-chain fatty acids
孟然; 杨孜; 王海玲; 王雁玲; 李凤秋; 韩怡炜
关键词长链游离脂肪酸 基因甲基化 长链三羟基酰基辅酶a脱氢酶 滋养层细胞 Long-chain Free Fatty Acids Methylation Lchad Trophoblast Cells
刊名中华医学杂志
2015
DOI10.3760/cma.j.issn.0376-2491.2015.29.013
95期:29页:2387-2392
收录类别中国科技核心期刊 ; 中文核心期刊 ; CSCD
文章类型Journal Article
摘要目的 探讨长链游离脂肪酸对胎盘滋养细胞线粒体β氧化循环长链三羟基酰基辅酶A脱氢酶(LCHAD)基因启动子区甲基化程度影响,以及对甲基化修饰的时间效应.方法 体外培养原代人绒毛滋养细胞及HTR-8/SVneo永久性人滋养细胞,研究组采用长链游离脂肪酸孵育滋养细胞(LC-FFA组),并以短链游离脂肪酸(SC-FFA组)和中链游离脂肪酸(MC-FFA组)作为不同链长游离脂肪酸对照组,空白对照组无脂肪酸(F-FFA组).分别在孵育24、48及72 h收集细胞并提取DNA.在LCHAD基因转录起始位点上游2 000 bp区域内序列预测CpG岛位置,设计甲基化检测位点和引物.采用Sequenom MassArray甲基化DNA定量分析平台检测不同组CpG岛各CpG位点的甲基化程度后进行各甲基化位点和CpG岛的统计学分析.结果 (1)在LCHAD基因转录起始位点上游2 000bp区域内的17个CpG位点中获得11个位点(65%)的甲基化程度,8个为单独位点,3个为复合位点(-984、-960、-899、-853、-811、-796、-774、-727、-615、-595、-579).在不同研究组中每个位点的甲基化变化程度各不同,其中-899位点变化最明显.(2)LCHAD基因启动子区CpG岛甲基化程度变化分析:①长、中链游离脂肪酸对LCHAD基因启动子区CpG岛甲基化程度影响伴随时间的延长呈现升高趋势:LC-FFA组孵育72 h(0.55±0.08)比孵育48 h(0.35±0.12)及24 h(0.31±0.04)均明显升高(P<0.05),48 h虽比24 h升高,但差异无统计学意义(P>0.05).MC-FFA组孵育72 h(0.44±0.05)比孵育24 h(0.31±0.04)明显升高(P<0.05).②不同链长游离脂肪酸在不同作用时间影响LCHAD基因启动子区CpG岛甲基化程度比较:LC-FFA组孵育72 h的甲基化程度比MC-FFA、SC-FFA组及F-FFA组的72、48及24 h均明显升高(P<0.05).(3)作用72 h游离脂肪酸对LCHAD基因启动子区11个CpG位点甲基化程度影响比较:在LC-FFA组和MC-FFA组-899位点甲基化程度明显高于其他位点(P<0.05).(4)长链游离脂肪酸对LCHAD基因启动子区-899位点甲基化程度影响伴随时间的延长呈现升高趋势(P<0.05).LC-FFA组孵育72 h比MC-FFA、SC-FFA组及F-FFA组的72、48及24 h均明显升高(P<0.05).结论 长链游离脂肪酸比中链和短链游离脂肪酸呈现较大的滋养细胞LCHAD基因启动子区甲基化的修饰作用,并伴随作用时间延长表现出明显的时间依赖效应;甲基化程度改变主要发生在LCHAD基因启动子区-899位点. Objective To explore the methylation level of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) gene promoter region in mitochondria of trophoblasts incubated with long-chain fatty acids and the time-effect of methylation modification.Methods Primary human trophoblast cells and HTR8/Svneo cells were incubated with free fatty acids of various lengths.Long-chain free fatty acids (LC-FFA) was experimental group,short-chain fatty acids (SC-FFA) and medium-chain fatty acids (MC-FFA) were control groups,and blank control was without free fatty acid.Collecting cells and extract DNA at 24,48 and 72 h incubation respectively.Predicted CpG island location access to 2 000 bp DNA sequences upstream of the transcription start site of LCHAD gene.We designed methylation detection sites and primer originally.Methylation of CpG sites in LCHAD gene promoter region were detected by MassARRAY and analyzed statistically.Results (1) We detected methylation of 65% (11/17) CpG sites,including 8 single sites and 3 composite sites,in ampliconic sequences.These CpG sites were at positions:-984,-960,-899,-853,-811,-796,-774,-727,-615,-595,-579,respectively.In different groups methylation level and changes of every site showed differences with the most significant changes at -899 site.(2) Methylation of CpG island in LCHAD gene promoter region:① Methylation of CpG island in LC-FFA and MC-FFA groups showed rising trend with the time:Methylation level of LC-FFA group at 72 h (0.55 ± 0.08) was significantly higher than that of 48 h (0.35 ± 0.12) and 24 h (0.31 ± 0.04) (P < 0.05).Methylation level of MC-FFA group at 72 h (0.44 ± 0.05) was significantly higher than that of 24 h (0.31 ± 0.04) (P < 0.05).② Methylation of CpG island in LCHAD gene promoter region in different groups at different times:Methylation level of LC-FFA group at 72 h was significantly higher than that of the other two groups at 72,48 and 24 h (P < 0.05).(3) Methylation of 11 CpG sites in LCHAD gene promoter region in different groups at 72 h:Methylation level of-899 site in LC-FFA and MC-FFA groups were significantly higher than that of other sites (P < 0.05).(4) Methylation level of-899 site in LCHAD gene promoter region in LC-FFA group showed rising trend with the time:(72 h (0.34 ±0.15),48 h (0.14 ± 0.05) and 24 h (0.10 ±0.02),P <0.05).Methylation level of LC-FFA group at 72 h was significantly higher than that of the other two groups at 72,48 and 24 h (P < 0.05).Conclusions Methylation modification effect on LCHAD gene promoter region in trophoblast cells incubated with long-chain fatty acids is more significant than with medium-chain and short-chain fatty acids and shows obvious time-effect as incubation time prolonged.The changes at-899 site dominate the degree of methylation in LCHAD gene promoter region.
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文献类型期刊论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/69841
专题北京大学第三临床医学院_妇产科
作者单位1.100191,北京大学第三医院妇产科
2.中国科学院动物研究所计划生育生殖生物学国家重点实验室
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孟然,杨孜,王海玲,等. 长链游离脂肪酸对滋养细胞线粒体三羟基酰基辅酶A脱氢酶基因启动子区甲基化程度的影响[J]. 中华医学杂志,2015,95(29):2387-2392.
APA 孟然,杨孜,王海玲,王雁玲,李凤秋,&韩怡炜.(2015).长链游离脂肪酸对滋养细胞线粒体三羟基酰基辅酶A脱氢酶基因启动子区甲基化程度的影响.中华医学杂志,95(29),2387-2392.
MLA 孟然,et al."长链游离脂肪酸对滋养细胞线粒体三羟基酰基辅酶A脱氢酶基因启动子区甲基化程度的影响".中华医学杂志 95.29(2015):2387-2392.
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