TCDD诱导的胎鼠腭裂发生过程中TGF—β3、Dnmts启动子区CpG岛甲基化状态研究

Experimental research on methylation status of CpG islands in promoter region of TGF-β3 and Dnmts during TCDD-induced fetal palatogenisis

目的研究2，3，7，8-四氯二苯二噁英（2，3，7，8-tetrachlrodibenzo-p—dioxin，TCDD）诱导的胎鼠腭发育过程中转化生长因子β3（transforming growth factor beta3，TGF-β3）、DNA甲基转移酶（DNA methyltransferases，Dnmts）基因启动子区CpG岛甲基化状态与TGF—β1、Dnmts表达量的相关性。方法将18只C57BL／6J孕鼠完全随机分为TCDD组和对照组，每组各9只。TCDD组于妊娠第10．5天（gestation day，GD10．5）给予28μg／kg的TCDD口服，对照组给予等体重体积的玉米油口服。分别于GD13．5、14．5、15．5处死孕鼠，收集2组胎鼠腭突组织。通过试剂盒进行基因组DNA提取、亚硫酸氢盐转化、甲基化特异性PCR，将PCR产物进行琼脂糖凝胶电泳，分析2组各时间点腭组织表达的TGF-β3、Dnmts启动子CpG岛甲基化状态。应用IBMSPSS20．0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2组各时间点TGF—β3启动子区CpG岛均处于低甲基化水平，TCDD组和对照组GD13．5、GD14．5、GD15．5甲基化程度分别为（8．6±0．8）％和（8．7±0．8）％、（11．5±1．4）％和（11．7±1．0）％、（12．0±0．7）％和（12．1±0．5）％，相同时间点TCDD组与对照组间差异无统计学意义（P〉0．05）。Dnmt1启动子区CpG岛均处于高甲基化水平，TCDD组和对照组GD13．5、GD14．5、GD15．5甲基化程度分别为（73．9±1．1）％和（72．6±0．8）％、（70．8±1．7）％和（70．7±1．0）％、（69．4±2．2）％和（69．7±0．5）％，相同时间点TCDD组与对照组间差异亦无统计学意义（P〉0．05）。TCDD组Dnmt3a启动子区CpG岛1的甲基化水平在GD13．5和GD15．5高于对照组，分别为（21．9±1．1）％和（8．1±0．6）％（P〈0．01）、（43．4±0．4）％和（32．9±0．7）％（P〈0．01），在GD14．5低于对照组，分别为（33．2±0．5）％和（42．9±0．3）％（P〈0．01）。对照组Dnmt3a启动子区CpG岛2的甲基化水平在各时间点均高于TCDD组，GD13．5、GD14．5、GD15．5甲基化程度分别为（82．0±0．7）％和（32．3±0．6）％（P〈0．01）、（62．7±1．0）％和（25．5±1．4）％（P〈0．01）、（47．2±0．4）％和（30．3±1．4）％（P〈0．01）。结论TCDD诱导的胎鼠腭发育期间Dnmt3a启动子区接近第1外显子处CpG岛2的低甲基化水平，可能是Dnmt3a基因在GD13．5高表达的原因，并因此诱发胎鼠腭裂的基因组呈高甲基化状态。TGF—β3和Dnmtl启动子区CpG岛甲基化状态与TCDD作用无关。

Objective To investigate the correlation between CpG islands methylation statuses of TGF-β3, Dnmts and their expression during TCDD-induced mouse embryonic palatal development. Methods Eithtteen pregnant C57BL/6J mice were randomly divided into 2 groups： the control group（n = 9） and TCDD-exposure group（n = 9）. At 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 at GD13.5, GD14.5, GD15.5, fetal palates were collected. CpG island methylation statuses were analysed by methylation specific polymerase chain reaction （MSP）. IBM SPSS 20.0 software was applied for statistical analysis. Kolmogorov-Smirnov test was used for normal distribution check, and the distributions were normal. Independent t-test was carried out between two groups. P 〈 0.05 was eonsidered statistically significant. Results CpG island in promoter region of gene TGF-β3 were all at low methylation level at all GDs of both groups, there were no differences at same GD between two groups [GD13.5：（8.6±0.8）% vs （8.7 ±0.8）%, P〉0.05;GD14.5：（11.5 ±1.4）%vs （11.7±1.0）%, P〉0.05;GD15.5： （12.0±0.7）% vs （12.1 ±0.5）%, P〉0.05%. CpG island in promoter region of gene Dnmtl were all highly methylated with no differenees showed at same GD between two groups [GD13.5：（73.9±1.1）%vs （72.6±0.8）%,P〉0. 05;GD14.5：（70.8±1.7）% vs （70.7±1.0）%, P〉0.05;GD15.5：（69.4±2.2）% vs （69.7±0.5）%,P〉0.05].The methylation level of CpG island 1 in promoter region of gene Dnmt3a in TCDD group was higher than that in eontrol group at GD13.5 and GD15.5 [（21.9±1.1）% vs （8.1 ±0.6）% ,P〈0.01,（43.4±0.4）% vs（32.9±0.7）%,P〈0.01], while lower at GD14.5[（33.2±0.5）% vs （42.9 ±0.3）%,P〈0.01]. The methylation level of CpG island 2 in promoter region of gene Dnmt3a in control group was higher than that in TCDD group at all GDs [GD13.5：（82.0±0.7）% vs （32.3±0.6）%,P〈0.01;GD14.5：（62.7 ±1.0）%vs （25.5 ±1.4）%, P〈0.01;GD15.5：（47,2±0.4）% vs（30.3±1.4）%,P〈0.01]. Conclusions Low methylation level of CpG island 2 which is close to the first exon in promoter region of gene Dnm3a may be the cause of highly expressed Dnmt3a mRNA at GD13.5 during mice palatogenesis induced by TCDD, thus the global DNA methylation is extremely high to induce cleft palate. TCDD-treatment doesn＇t influence the CpG methylation statuses in promoter region of TGF-β3 and Dnmtl.