启动子区域甲基化状态对THP-1细胞肿瘤坏死因子α分泌的影响  

Effects of methylation status of gene promoter on secretion of tumor necrosis factor alpha in THP-1 cells

作　　者：郭尧[1] 杨智勇[1] 王春友[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院胰腺病研究所,湖北武汉430022

出　　处：《中国病理生理杂志》2011年第6期1206-1209,共4页Chinese Journal of Pathophysiology

摘　　要：目的:探讨肿瘤坏死因子α(TNF-α)基因启动子区域散在CpG双核苷酸结构甲基化状态对蛋白分泌和基因表达的影响。方法:检测人单核细胞系THP-1细胞在脂多糖(LPS)刺激状态下,不同时点TNF-α的表达和其基因启动子区域的甲基化状态。采用ELISA法测定细胞培养液上清TNF-α水平;采用重亚硫酸盐转化基因测序法测定DNA甲基化状态。结果:受到LPS刺激后,THP-1细胞迅速产生TNF-α,在4 h达到高峰后快速下降。TNF-α基因启动子-344 bp到转录起始位点(TSS)区域内,存在11个散在CpG双核苷酸结构。未受到LPS刺激时均处于甲基化状态;刺激4 h后,有4个处于甲基化状态;刺激8 h后,有9个处于甲基化状态。未刺激与刺激4 h之间,刺激4 h与刺激8 h之间,甲基化率比较均有显著差异(P<0.01,P<0.05)。结论:未受到LPS刺激时,THP-1细胞TNF-α基因启动子区域甲基化水平较高;LPS刺激后,其该区域甲基化水平降低。该变化与TNF-α表达分泌相关。AIM：To investigate the correlation between the expression and secretion of tumor necrosis factor alpha（TNF-α） and the methylation status of TNF-α promoter.METHODS： The secretion level of TNF-α in THP-1 cells stimulated with lipopolysaccharide （LPS） for different time was examined by enzyme-linked immunosorbent assay（ELISA）.The methylation status of TNF-α promoter was measured with bisulfite sequencing analysis.RESULTS： In unstimulated THP-1 cells,the secretion level of TNF-α was very low,and all the 11 CpG dinucleotides in gene promoter ranging from-344 bp to transcription start site（TSS） were completely methylated.In 4 h LPS-stimulated THP-1 cells, the secretion level of TNF-α reached peak concentration,while CpG dinucleotides in gene promoter were extensively demethylated and only 4 in 11 were methylated.In 8 h LPS-stimulated THP-1 cells,the secretion level of TNF-α dropped from the high concentration,and 9 CpG dinucleotides in 11 in gene promoter were methylated.The percentages of methylation between unstimulated THP-1 cells and 4 h LPS-stimulated THP-1 cells were statistically different（P0.01）.The percentages of methylation between 4 h and 8 h LPS-stimulated THP-1 cells were also statistically different（P0.05）.CONCLUSION： The methylation status of TNF-α promoter shows an association with the expression and secretion of TNF-α.

关 键 词：肿瘤坏死因子 DNA甲基化 脂多糖类 THP-1细胞 

分 类 号：R363[医药卫生—病理学]