隐睾发生过程中DNA甲基化状态和甲基化转移酶表达的实验研究

DNA methylation changes and DNA methyltransferases＇ expression during cryptorchidism formation induced by di-2-（ethylhexyl） phthalate in mice.

目的研究环境内分泌干扰物邻苯二甲酸二（2-乙基已基）酯[Di—（2-ethylhexy）phthalate，DEHP]作用于孕鼠后，胎鼠睾丸组织基因组DNA甲基化水平的变化及3种DNA甲基化转移酶的表达情况；探讨DEHP所致表观遗传修饰改变在睾丸下降过程中的作用。方法妊娠昆明小鼠（KMmice）随机分为3组，即正常对照组、玉米油对照组和DEHP实验组，每组15只。玉米油对照组和DEHP实验组自妊娠第12．5天到妊娠第18．5天分别持续经口予以玉米油和DEHP，用量按每日500mg／kg，正常对照组不予灌药。采用高效液相色谱仪检测基因组DNA甲基化水平，实时荧光定量PCR检测3种DNA甲基化转移酶的表达量。结果正常对照组和玉米油对照组DNA甲基化水平和3种甲基化转移酶的表达均无统计学意义（P〉0．05）：DEHP实验组DNA甲基化水平比对照组增高约10％。与对照组相比，差异有显著统计学意义（P〈0．05）；实验组3种甲基化转移酶的表达量均比对照组高，与对照组相比，差异有显著统计学意义（Dnmt1，P〈0．05；Dnmt3a，P〈0．01；Dnmt3b，P〈0．05）。结论环境内分泌干扰物DEHP通过影响3种甲基化转移酶的表达而导致基因组DNA甲基化水平升高，从而抑制睾丸下降过程中多种基因的表达，发生隐睾。DNA甲基化等表观遗传学修饰的改变可能是DEHP诱发隐睾的分子机制之一。

Objective To analyze epigenetic （specifically, DNA methylation） and DNA methyltransferases participation in the mechanisms of cryptorebidism only induced by maternal exposure to Di-2-（ethylhexyl） phthalate （DEHP） in mice. Methods KM miees were divided into 3 groups randomly： normal control group, corn oil group and DEHP-exposed group. Corn oil and DEHP [500 mg/ （kg· d）] were administrated respectively to the pregnant mice from gestation day 12.5 （GD12.5） to GD18.5. Testes were excised for analysis at GD19. High-performance liquid chromatography （HPLC） was performed to analyze DNA methylation status. The expression levels of the DNA methyhransferases were examined by Quantitative real-time PCR （qPCR）. Results Using HPLC, at GD19, the status of DNA methylation in global DNA was increased significantly in DEHP-treated groups compared with the controls （P 〈 0.05）. There were also significant changes in the expression of DNA methyhransferases （Dnmtl ,P 〈 0.05;Dnmt3a,P 〈 0.01 ;Dumt3b,P 〈 0.05）. Conclusions These results indicate that changes in DNA methylation may play an important role in the manifestation of cryptorchidism only caused by environmental factors such as maternal exposure to DEHP.