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目的:建立孕期苯巴比妥暴露所致的IUGR模型,观察胎肾上腺胆固醇供应关键基因——清道夫受体B类I型(scavenger receptor class B type I,SR-BI)与乙酰乙酰辅酶A合成酶(acetoacetyl-CoA synthetase,AACS)的表达与DNA甲基化修饰改变,验证此关键基因的DNA高甲基化作为胎肾上腺化合物发育毒性评价标志物的可能性。方法:健康Wistar孕鼠于受孕第7~17天灌胃给予50 mg·kg-1·d-1苯巴比妥或等量生理盐水,孕第17天处死,记录胎鼠体质量与胎盘重量,计算宫内发育迟缓(intrauterine growth retardation,IUGR)发生率。收集胎肾上腺用于透射电镜、荧光实时定量反转录PCR(reverse-transcription PCR,RTPCR)及亚硫酸氢盐测序PCR(bisulfite sequencing PCR,BSP)检测。结果:与对照组相比,苯巴比妥暴露组胎鼠体质量与胎盘重量均降低(P<0.01),IUGR发生率升高(P<0.01);透射电镜结果显示,胎肾上腺皮质细胞表现出线粒体水肿、胞浆内空泡等现象;RT-PCR检测结果显示,SR-BI与AACS表达降低(P<0.05,P<0.01);BSP结果显示,SR-BI与AACS近端启动子区DNA甲基化频率升高(P<0.01)。结论:孕期苯巴比妥暴露所致IUGR模型中,胎肾上腺组织结构受损,胆固醇供应关键基因SR-BI与AACS的mRNA表达改变,SR-BI与AACS的DNA高甲基化可作为潜在的化合物发育毒性评价标志物。
OBJECTIVE: To establish a model of IUGR induced by phenobarbital exposure during pregnancy and observe the synthesis of scavenger receptor class B type I (SR-BI) and acetoacetyl-CoA The expression of acetoacetyl-CoA synthetase (AACS) and the DNA methylation modification were changed to verify the possibility of DNA hypermethylation of this key gene as a marker of fetal adrenal developmental toxicity. Methods: Healthy Wistar pregnant rats were orally administered with 50 mg · kg-1 · d-1 phenobarbital or an equal volume of saline on the 7th to 17th day after pregnancy. The pregnant rats were sacrificed on the 17th day of pregnancy. The body weight and placental weight , Calculate intrauterine growth retardation (IUGR) incidence. The fetal adrenal glands were collected for transmission electron microscopy, reverse transcription PCR (RTPCR) and bisulfite sequencing PCR (BSP). Results: Compared with the control group, the body weight and placental weight of the fetus with phenobarbital exposure decreased (P <0.01) and the incidence of IUGR increased (P <0.01). Transmission electron microscopy showed that fetal adrenal cortex The results of RT-PCR showed that the expression of SR-BI and AACS was decreased (P <0.05, P <0.01). The BSP results showed that SR-BI and AACS proximal promoter region DNA methylation frequency increased (P <0.01). CONCLUSIONS: In IUGR model induced by phenobarbital exposure during pregnancy, fetal adrenal tissue structure is impaired. The mRNA expression of key genes SR-BI and AACS of cholesterol supply is changed. DNA hypermethylation of SR-BI and AACS can be used as potential compounds Toxicity evaluation marker.