短期暴露于纳米SiO_2对HaCaT细胞基因组DNA甲基化的影响 |
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引用本文: | 龚春梅,杨淋清,陶功华,刘庆成,刘建军,庄志雄.短期暴露于纳米SiO_2对HaCaT细胞基因组DNA甲基化的影响[J].卫生研究,2013,42(2). |
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作者姓名: | 龚春梅 杨淋清 陶功华 刘庆成 刘建军 庄志雄 |
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作者单位: | 1. 深圳市慢性病防治中心,深圳,518020 2. 深圳市疾病预防控制中心 |
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基金项目: | 国家自然科学基金项目,深圳市重点项目 |
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摘 要: | 目的探讨纳米SiO2对体外培养细胞基因组总体DNA甲基化水平的影响。方法分别以2.5、5、10μg/ml纳米SiO2溶液和10μg/ml微米级SiO2处理人皮肤表皮细胞系(HaCaT)24h;以3μmol/L DNA甲基化转移酶抑制剂5-脱氧杂氮胞苷(DAC)处理48 h的10μg/ml组为阳性对照,并设立溶剂对照组。应用高效毛细管电泳(HPCE)定量分析纳米SiO2处理细胞24h后,基因组DNA总体甲基化的变化,用Q-PCR和Western Blot方法检测甲基化相关蛋白mRNA和蛋白的表达变化,并用DNA甲基化转移酶活性试剂盒检测DNA甲基化转移酶的活性。结果 HPCE定量分析结果显示纳米SiO2可引起HaCaT细胞总体甲基化程度降低,呈剂量依赖性关系;与正常细胞相比,微米SiO2以及2.5、5和10μg/ml纳米SiO2组分别降低37.8%、43.9%、54.9%和52.8%,差异有显著性(P<0.05);对照组5-aza-dC处理后使HaCaT细胞甲基化程度减少27.3%。各组酶的蛋白表达与mRNA表达水平及DNMTs活性变化具有相同的变化趋势,经纳米SiO2处理的HaCaT细胞,DNMT1、DNMT3a及MBD2表达随着纳米SiO2剂量的上升而下降,DAC组表达水平最低。结论纳米SiO2能引起HaCaT细胞整体基因组DNA甲基化水平降低,可能与DNA甲基化转移酶的酶活性降低有关。
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关 键 词: | DNA甲基化 高效毛细管电泳(HPCE) 纳米SiO2 |
Genome DNA hypomethylation in HaCaT cells after short exposure to SiO2 nanoparticles |
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Abstract: | Objective To observe the effect of SiO2 nanoparticles on genome DNA methylation profile in cultured cells.Methods HaCaT cells were treated with nm-SiO2 at 2.5,5 and 10μg/ml and micro-SiO2 at 10μg/ml for 24h and DAC treatment was given at 10μg/ml group for 48h.The mC/(mC+C) percent was quantified by high performance capillary electrophoresis(HPCE) assay,and the expression level of mRNA and protein was detected by Real-time Q-PCR and westernblot assay.The activity of DNMTs was determined by DNA Methyltransferase Activity/Inhibition Assay Kit.Results HPCE assay showed that nm-SiO2-treated cells were decreased in some degree.An average proportion of methylated mC/(mC+C) was 4.82% in control,2.7% in 2.5μg/ml and 2.17% in 10μg/ml groups,while 3.1% in micro-SiO2 groups,which got the consistent downtrend of genome methylation level during increasing nm-SiO2 dose nanoparticles.The mRNA expression level for DNMT1 decreased gradually with increased dose of nm-SiO2 nanoparticles.The alterations at protein level were similar to those at the mRNA level.Conclusion Genomic DNA methylation levels were decreased in HaCaT cells after short term exposure to SiO2 nanoparticles. |
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Keywords: | DNA methylation high performance capillary electrophoresis SiO2 nanoparticles(nm-SiO2) HaCaT cell |
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