TCDD诱导胎鼠腭裂发生过程中DNA甲基化变化的实验研究

发布时间：2018-04-12 12:07

本文选题：腭裂 + 动物模型　；参考：《重庆医科大学》2017年硕士论文

【摘要】：第一部分TCDD诱导C57BL/6J胎鼠腭裂的最适剂量再探讨目的:观察不同剂量环境污染物2,3,7,8-四氯二苯二VA英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)诱导的C57BL/6J胎鼠腭部发育情况及死胎发生,探讨腭裂发生率和死胎发生率与TCDD剂量的相关性。方法:于合笼交配后第10.5天(GD10.5),将判断受孕并称重的C57BL/6J孕鼠分为不同剂量给药组给予0、8、18、28μg/kg的TCDD。观察孕鼠活动情况至GD17.5,处死孕鼠,剖腹取胎,观察死胎发生情况。于解剖显微镜下观察存活胚胎腭部发育情况。结果:对照组与实验组在孕鼠体重增加、每窝胚胎数上无差异(P0.05),各剂量组产生的腭裂表型无差异。对照组无腭裂及死胎发生。各剂量组腭裂发生率均显著高于对照组(P0.01);但TCDD18μg/kg和28μg/kg组间腭裂发生率无统计学差异(P0.05);仅TCDD28μg/kg组与更低剂量组及对照组的死胎发生率存在差异(P0.05)。结论:TCDD20μg/kg~30μg/kg剂量区间是诱导腭裂高发生率的平台区间,即为诱导C57BL/6J胎鼠腭裂高发生率的最适剂量。第二部分腭发育过程中基因组DNA甲基化的初步研究目的:研究TCDD作用于C57BL/6J孕鼠后,胎鼠腭组织基因组DNA甲基化水平变化及正向调控甲基化水平的DNA甲基转移酶(DNA methyltranferases,Dnmts)和负向调控甲基化水平的十-十一转位蛋白(Ten-Eleven Translocations,TETs)m RNA的表达情况,明确基因组DNA甲基化在腭发育过程中的作用。方法:C57BL/6J孕鼠共40只,随机分为TCDD组和对照组,每组20只。TCDD组于受孕后第10.5天(gestation day,GD10.5)以TCDD28μg/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、GD14.5、GD15.5、GD16.5、GD17.5处死孕鼠,收集各时间点的胎鼠腭组织,分别提取基因组DNA、总RNA。应用MethylampT.M.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Dnmt1、Dnmt3a、Dnmt3b以及TET1、TET2、TET3 m RNA的表达量。结果:TCDD组标准化甲基化率在GD13.5显著高于对照组组(P0.01);而在GD14.5、16.5均低于对照组(P0.05,P0.05)。在GD13.5、16.5,TCDD组Dnmt1 m RNA表达量高于对照组(P0.05,P0.01)。在GD13.5、16.5,TCDD组Dnmt3a m RNA表达量高于对照组(P0.05,P0.05)。在GD14.5,TCDD组Dnmt3b m RNA表达量显著高于对照组(P0.01)。在GD13.5,TCDD组TET3 m RNA表达量显著低于对照组(P0.01)。结论:胎鼠腭组织内可能存在复杂的DNA甲基化调控机制。由Dnmt1、Dnmt3a表达升高以及TET3表达降低引起的腭组织GD13.5基因组DNA甲基化水平的显著升高可能是TCDD致胎鼠腭发育障碍的表观遗传机制之一。第三部分TGF-β3、Dnmt1、Dnmt3a基因启动子区Cp G岛甲基化状态的实验研究目的:探讨TGF-β3、Dnmts基因启动子Cp G岛甲基化状态与TCDD所致胎鼠腭发育障碍的相关性。方法:将已知孕期的C57BL/6J孕鼠共18只,随机分为TCDD组和对照组。TCDD组于GD10.5给予28μg/kg的TCDD,对照组给予等体重体积的玉米油。分别于GD13.5、14.5、15.5处死孕鼠,收集各组胎鼠腭突组织。通过试剂盒提取组织DNA、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各组各时间点腭组织表达的TGF-β3、Dnmts启动子Cp G岛甲基化状态。结果:各组各时间点TGF-β3启动子区Cp G岛均处于低甲基化水平,相同时间点两组间无统计学差异(P0.05)。Dnmt1启动子区Cp G岛均处于高甲基化水平,相同时间点两组间亦无统计学差异(P0.05)。TCDD组Dnmt3a启动子区Cp G岛1的甲基化水平在GD13.5和GD15.5高于对照组(P0.01,P0.01),在GD14.5低于对照组(P0.01)。对照组Dnmt3a启动子区Cp G岛2的甲基化水平在各时间点均高于TCDD组(P0.01,P0.01,P0.01)。结论:TCDD诱导的胎鼠腭发育期间Dnmt3a启动子区接近第一外显子处Cp G岛低甲基化水平可能是Dnmt3a基因在GD13.5高表达的原因,并因此形成异常的基因组高甲基化状态,进而诱发胎鼠腭裂。
[Abstract]:The optimal dosage of the first part of the TCDD induced C57BL/6J rat palate of objective: To observe the different dosage of environmental pollutants 2,3,7,8- four chloro two benzene two VA (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) development and stillbirth induced C57BL/6J rat palate, to explore the correlation between the rate and the incidence of TCDD and dose of cleft palate. Methods: in mated 10.5 day (GD10.5), will determine the pregnancy and pregnant C57BL/6J rats weighing different dosage group were given 0,8,18,28 g/kg TCDD. to observe the activity of pregnant rats to GD17.5, pregnant mice were killed, caesarean section, to investigate the incidence of stillbirth. In under a dissecting microscope to observe the survival of embryonic palatal development. Results: control group and experimental group increased in pregnant rats body weight, the number of embryos per litter no difference (P0.05), no difference in all dose groups. The control group without cleft palate cleft palate phenotype and stillbirth. The incidence of cleft palate group were significantly higher than those in control group (P0.01); but TCDD18 g/kg and 28 g/kg group there was no significant difference between cleft palate (P0.05); only TCDD28 g/kg group and lower dose group and control group, the incidence of differences (P0.05). Conclusion: TCDD20 g/kg~30 g/kg dose interval is the high incidence of cleft palate induced by the platform interval, the optimal dosage of C57BL/6J fetal rats induced high incidence of cleft palate development. The second part objective to investigate the genomic DNA methylation process: To study the effect of TCDD on C57BL/6J rats after methyl palatal tissues of fetal rats by DNA methylation level and positive regulation of the methylation level of DNA transferase (DNA methyltranferases, Dnmts) and negatively regulates the methylation level of ten - eleven protein translocation (Ten-Eleven Translocations, TETs) expression of M RNA, clear the genomic DNA methylation process in the development of palate The role of C57BL/6J in pregnant rats. Methods: a total of 40 rats were randomly divided into TCDD group and control group, 20 rats in each group.TCDD after conception 10.5 days (gestation day, GD10.5) with TCDD28 g/kg administered once; the control group was injected with the same amount of disposable corn oil gavage; respectively in GD13.5, GD14.5, GD15.5 GD16.5, GD17.5, pregnant rats were killed at each time point, collecting the palatal tissues of fetal rats, respectively from the genomic DNA of total RNA. using MethylampT.M. genomic DNA methylation assay kit to detect the methylation level of genomic DNA, real-time fluorescence quantitative PCR detection of Dnmt1, Dnmt3a, Dnmt3b, TET1, TET2, m RNA expression of TET3. Results group TCDD: Standard methylation rate in GD13.5 group was significantly higher than that of control group (P0.01); while the GD14.5,16.5 was lower than the control group (P0.05, P0.05). In GD13.5,16.5, TCDD Dnmt1 m RNA group was higher than that of control group (P0.05, P0.01). In GD13.5,16.5 group the expression of TCDD Dnmt3a m RNA The amount is higher than the control group (P0.05, P0.05). In GD14.5, Dnmt3b m RNA expression in TCDD group was significantly higher than that of the control group (P0.01). In GD13.5, TET3 m RNA expression in TCDD group was significantly lower than the control group (P0.01). Conclusion: there may be DNA methylation regulation of complex palate tissues of fetal rats. By Dnmt1, the expression of Dnmt3a increased and decreased expression of TET3 caused by the palatal tissue GD13.5 DNA methylation significantly increased may be one of the mechanisms of epigenetic TCDD induced mouse embryonic palatal developmental disorder. The third part of TGF- beta 3, Dnmt1, Dnmt3a gene objective to study the promoter region of Cp G island methylation status: To investigate TGF- beta 3, Dnmts gene relationship between promoter Cp methylation of the G and TCDD induced mouse embryonic palatal developmental disorder. Methods: C57BL/6J rats were only 18 known during pregnancy, were randomly divided into TCDD group and control group in.TCDD group GD10.5 given 28 g/kg TCDD, the control group was given The volume weight of corn oil. Pregnant mice were killed in GD13.5,14.5,15.5 respectively, were collected to mouse embryonic palatal tissue. Through tissue DNA extraction kit, bisulfite conversion, methylation specific PCR, PCR agarose gel electrophoresis analysis of the expression of each group at different time points of palate tissue TGF- beta 3, Dnmts promoter Cp the G Island methylation status. Results: each group at different time points of TGF- beta 3 promoter region Cp island of G are in the low level of methylation at the same time, no significant difference between the two groups (P0.05.Dnmt1) Cp in the promoter region G island methylation were at the high level, at the same time there was no statistical difference between the two groups (P0.05).TCDD group Dnmt3a launched 1 Cp promoter G island methylation level was higher than the control group in GD13.5 and GD15.5 (P0.01, P0.01), in GD14.5 was lower than the control group (P0.01). The control group, the methylation level of Dnmt3a promoter Cp island of G 2 were higher than that of group TCDD at each time point (P0.01 , P0.01, P0.01). Conclusion: TCDD induced mouse embryonic palatal development during the Dnmt3a promoter region close to the first exon of Cp G island methylation level may be the reason for the high expression of Dnmt3a gene in GD13.5 genome, and thus the formation of high methylation abnormalities, and induce fetal cleft palate.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782.22

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