摘要
目的 研究2,3,7,8-四氯二苯二噁英（2,3,7,8-tetrachlrodibenzo-p-dioxin,TCDD）作用于C57BL/6J孕鼠后,胎鼠腭组织基因组DNA甲基化水平变化及DNA甲基转移酶的表达情况.方法 C57BL/6J孕鼠共40只,完全随机分为TCDD组和对照组,每组20只.TCDD组于妊娠第10.5天（GD10.5）以TCDD28 μg/kg一次性灌胃,对照组以等量玉米油一次性灌胃;分别于GD13.5、14.5、15.5、16.5、17.5处死孕鼠,采集各时间点的胎鼠腭组织,分别提取基因组DNA、总RNA.应用MethylampTM基因组DNA甲基化定量试剂盒检测基因组DNA甲基化水平,实时荧光定量PCR检测DNA甲基转移酶Dnmt1、Dnmt3a和Dnmt3b mRNA的表达量.应用IBM SPSS 20.0软件进行统计分析,两样本间均数比较均采用独立样本t检验,所有结果作K-S检验均符合正态分布,方差不齐者采用校正t检验,P ＜0.05表示差异有统计学意义.结果 GD13.5、14.5和16.5胎鼠腭组织DNA甲基化水平：对照组分别为33.42％±6.78％、30.12％±3.92％和36.45％±3.27％,TCDD组分别为49.52％ ±4.03％、24.10％ ±2.29％和32.77％±0.98％.GD13.5,TCDD组DNA甲基化水平显著高于对照组组（P＜0.01）;而在GD14.5、16.5均低于对照组（P＜0.05）.GD13.5、16.5胎鼠腭组织Dnmt1 mRNA表达量：对照组分别为1.01 ±0.10和0.81 ±0.01,TCDD组分别为1.28±0.11和1.04±0.05.在GD13.5、16.5,TCDD组Dnmt1 mRNA表达量高于对照组（P ＜0.05,P＜0.01）.GD13.5、16.5胎鼠腭组织Dnmt3a mRNA表达量：对照组分别为0.81±0.02和0.96±0.06,TCDD组分别为1.15 ±0.17和1.11 ±0.06.在GD13.5、16.5,TCDD组Dnmt3a mRNA表达量高于对照组（P ＜0.05,P ＜0.05）.GD14.5,对照组Dnmt3b mRNA表达量为0.72±0.06,TCDD组为0.97 ±0.06,TCDD组显著高于对照组（P＜0.01）.结论 胎鼠腭组织内可能存在复杂的DNA甲基化调控机制,Dnmt1、Dnmt3a表达升高引起腭组织基因组DNA甲基化水平在GD13.5时显著升高,可能是TCDD导致胎鼠腭发育障碍的表观遗传机制之一.
Objective To investigate global DNA methylation and DNA methyhransferases participation in the mechanism of cleft palate induced by maternal exposure to 2,3,7,8-tetrachlrodibenzo-p-dioxin （TCDD）in mice.Methods 40 pregnant C57BL/6J mice were randomly divided into 2 groups：the control group（n =20） and TCDD-exposure group（n =20）.On gestation day 10.5 （GD10.5）,the mice in TCDD-group were orally administrated with TCDD 28 μg/kg,while the mice in the control group received equivalent corn oil.The pregnant mice were sacrificed on GD13.5,GD14.5,GD15.5,GD16.5,GD17.5,fetal palates were collected for analysis.Global DNA methylation levels were detected by MethylampTM Global DNA Methylation Quantification Ultra Kit through an ELISA-like reaction.The expression levels of DNA methyltransferases were examined by quantitative real-time PC R（q-PCR）.IBM SPSS 20.0 software was applied for statistical analysis.Kolmogorov-Smirnov test was used for normal distribution check,and the distribution was normal.Independent t-test was carried out among two groups.P ＜ 0.05 was considered statistically significant.Results The global DNA methylation level in TCDD-exposure group was significantly higher than that in control group on GD13.5 （49.52％ ±4.03％ vs 33.42％ ± 6.78％,P ＜ 0.01）,whilelower on GD14.5 （24.10％ ±2.29％ vs 30.12％ ±3.92％,P ＜0.05） and on GD16.5 （32.77％ ±0.98％ vs 36.45％ ± 3.27％,P ＜ 0.05）.The expression level of Dnmt1 mRNA in TCDD-exposure group was higher than that in control group on GD13.5（1.28±0.11 vs 1.01 ±0.10,P＜0.05） and on GD16.5（1.04 ±0.05 vs 0.81 ±0.01,P ＜0.01）.The expression level of Dnmt3a mRNA in TCDD-exposure group was higher than that in control group on GD13.5 （1.15 ±0.17 vs 0.81 ±0.02,P ＜0.05）and on GD16.5 （1.11 ± 0.06 vs 0.96 ± 0.06,P ＜ 0.05）.The expression level of Dnmt3b mRNA in TCDD-exposure group was higher than that in control group on GD14.5（0.97 ±0.06 vs 0.72 ±0.06,P ＜0.01）.Conclusions It is supposed
出处
《中华整形外科杂志》
CAS
CSCD
北大核心
2016年第5期372-377,共6页
Chinese Journal of Plastic Surgery
基金
国家自然科学基金青年基金（N081202167）
重庆市渝中区科技计划项目（20130121）
国家临床重点专科建设项目：小儿外科学[国卫办医函（2013）544]
