妊娠早期高雌激素暴露对子代甲状腺激素的影响及其表观遗传机制研究

发布时间：2018-09-18 18:15

【摘要】：第一部分妊娠早期高雌激素暴露子代的随访研究目的：研究妊娠早期高雌激素环境暴露的子代的甲状腺功能状况,比较分析母亲妊娠时期的雌激素水平与甲状腺激素水平的相关性,明确宫内高雌环境所致成年期疾病的发病风险。材料和方法：对949名3-10岁的儿童甲状腺功能指标(T3,FT3,T4,FT4和TSH)进行了横断面研究,其中包括新鲜胚胎移植周期出生的IVF子代(Fresh ET组)357名,冷冻胚胎移植周期出生的IVF子代(Frozen ET组)212名,以及自然妊娠出生子代(NC组)380名。随访的指标包括子代的基本情况,其母亲妊娠时期的情况,以及子代的血压、心率、甲状腺激素、血常规等指标。通过病案系统回顾性查阅了2012年10月至2013年1月在浙江大学医学院附属妇产科医院进行辅助生殖治疗妊娠以及自然妊娠约4-12周时母亲外周血(单胎妊娠)的雌激素水平。此外,我们还检测了183名新生儿脐带血的甲状腺激素水平,其中包括Fresh ET组55名,Frozen ET组48名以及NC组80名,通过病案系统回顾性查阅了其母亲HCG日的雌激素水平。结果：排除出生体重、出生身长、孕周、性别、月龄、体重指数以及母亲妊娠年龄等干扰因素后,自然妊娠组、新鲜胚胎移植组和冷冻胚胎移植组子代的血常规指标、血压、心率无显著性差异,而子代甲状腺激素水平出现改变。Frozen ET组选取的均是自然周期进行冻融胚胎移植而出生的IVF子代,其母体雌激素水平为生理水平,与NC组没有显著差异,而Fresh ET组在孕早期各个时间点的母体雌激素水平远高于Frozen ET组和NC组。我们发现,Fresh ET组的T4,FT4和TSH水平显著高于NC组,Frozen ET组由于避开了宫内高雌激素环境,除FT4水平介Fresh ET组和NC组之外,T4和TSH水平跟NC组相比均没有显著性差异。同时,相比于NC组,Fresh ET组中FT4和TSH超过正常值上限的比例明显增加,提示发生甲状腺疾病的风险增加。在脐血中我们也发现,相比于对照组,Fresh ET组的T4,FT4和TSH显著增高,而Frozen ET组的T4和TSH接近于对照组。进一步分析发现通过Fresh ET出生的新生儿T4、FT4水平和母体hCG日的雌激素水平呈显著正相关。结论：促排卵治疗的孕妇在妊娠早期处于一种超生理范围的高雌激素状态,是研究宫内高雌激素暴露的优选模型。在胚胎发育过程中,超生理浓度的雌二醇水平会增加IVF子代甲状腺功能异常的风险。冰冻胚胎移植由于避开了宫内高雌激素环境,可以降低其出生子代甲状腺疾病风险。第二部分宫内高雌环境出生子代小鼠甲状腺功能及甲基化状况目的：研究宫内高雌环境出生子代小鼠的生长发育及甲状腺功能状况,明确宫内高雌环境所致成年期疾病的发病风险。材料和方法：建立妊娠期宫内高雌小鼠模型,雌激素给药组小鼠在妊娠第5-11天给予戊酸雌二醇,并收集妊娠第18天,出生后3周和8周的甲状腺、血液等标本。检测对照组(C)与高雌出生子代出生体重、3周龄及8周龄体重；放免法检测出生子代甲状腺激素水平；ELISA试剂盒检测给药后孕鼠体内雌激素水平；HE染色观察甲状腺形态；荧光定量PCR和免疫组化检测相关基因表达；亚硫酸氢盐测序检测pax8启动子区域CpG岛甲基化水平；体外研究17β-雌二醇对甲状腺滤泡细胞增殖的影响以及甲状腺转录因子pax8和甲基转移酶Dnmt 3a的表达调控。结果：妊娠时间和每窝出生仔数各组没有显著差异,但高雌组的妊娠率明显降低。此外高雌剂量组出生的子代雌鼠的体重在0-5周明显低于对照组,雄鼠跟对照组相比没有显著性差异。高雌剂量组出生的子代雌鼠在3周和8周时,T4和FT4显著高于对照组；高雌剂量组出生的子代雄鼠在3周时T4显著高于对照组,8周时跟对照组相比,没有显著性差异。荧光定量PCR和免疫组化结果显示,高雌组子代的Pax8和Tpo表达明显上调。进一步研究发现在高雌给药组出生的子代小鼠中,pax8基因启动子区域CpG岛甲基化水平明显低于对照组。同时,Dnmt 3a和Mbd1基因的表达要明显低于对照组。此外,在体外试验中,雌激素能够浓度依赖性地促进甲状腺滤泡细胞系的增殖,以及上调pax8基因和下调Dnmt3a基因。结论：在孕早期胎儿甲状腺发育的敏感时期,高浓度的雌激素暴露,有可能会改变甲状腺功能相关基因的甲基化状态,进而使得出生子代出现甲状腺功能的异常。第三部分长链非编码RNA在高雌激素暴露的甲状腺细胞中表观遗传调控的研究目的：研究高雌激素暴露对甲状腺滤泡细胞长链非编码RNA (lncRNA)表达谱的影响。筛选与甲状腺功能相关的lncRNA,验证其生物学功能并探讨相关分子机制,评估lncRNA在高雌激素致甲状腺功能异常发生发展中的作用,明确可以预测宫内高雌所致成年期疾病发生的长链非编码RNA。材料和方法：选取雌激素处理组和对照组甲状腺滤泡细胞各3例,进行lncRNAs表达芯片分析。采用实时定量PCR方法在雌激素处理的甲状腺滤泡细胞系中验证差异明显的6个lncRNAs。利用生物信息学对芯片结果进行分析,筛选目标lncRNA,并在nthy ori 3-1细胞上采用实时定量PCR方法验证雌激素处理后其表达的变化。应用小于扰RNA特异性降低lncDSG1的表达后,观察mdm2及甲状腺细胞增殖的变化。结果：我们筛选得到在高雌暴露的甲状腺滤泡细胞中差异表达的188个lncRNAs,其中上调表达的lncRNAs111个,下调表达的lncRNAs78个。我们在nthy ori 3-1细胞中验证了6个差异显著的lncRNAs,与芯片的结果一致,证实了芯片结果的可靠性。CNC网络图提示lnc DSG1可能影响甲状腺功能。进一步细胞实验也证实雌激素可以上调lnc DSG1和MDM2基因的表达,而加入干扰lnc DSG1后可以下调MDM2的表达并抑制甲状腺滤泡细胞的增殖。结论：高雌激素暴露的环境可导致lncRNAs表达谱的改变,雌激素上调lncRNADSG1的表达,促进MDM2的表达。而加入干扰lncRNA DSG1下调MDM2的表达并抑制甲状腺滤泡细胞的增殖。lncRNA DSG1可能参与了高雌激素暴露致甲状腺细胞功能异常的表观遗传学调控。
[Abstract]:Objective: To study the thyroid function of the offspring exposed to high estrogen in early pregnancy, to compare and analyze the relationship between estrogen level and thyroid hormone level during pregnancy, and to determine the risk of adult diseases caused by high estrogen in utero. Materials and Methods: A cross-sectional study of thyroid function parameters (T3, FT3, T4, FT4 and TSH) was carried out in 949 children aged 3-10 years, including 357 IVF offspring (Fresh ET group) born during the fresh embryo transfer cycle, 212 IVF offspring (Frozen ET group) born during the frozen embryo transfer cycle, and 380 natural pregnant offspring (NC group). Indicators included the basic conditions of the offspring, the pregnant period of the mother, blood pressure, heart rate, thyroid hormone, blood routine and other indicators of the offspring. Estrogen levels in maternal peripheral blood (single pregnancy) were also measured in 183 neonates, including 55 neonates in Fresh ET group, 48 in Frozen ET group and 80 in NC group. The maternal HCG day estrogen levels were retrospectively reviewed through a medical record system. Results: Birth weight, birth length, pregnancy were excluded. There were no significant differences in blood routine parameters, blood pressure and heart rate among the natural pregnancy group, fresh embryo transfer group and frozen embryo transfer group, but thyroid hormone levels were changed in the offspring. Frozen ET group was selected for frozen-thawed embryo transfer in the natural cycle. The maternal estrogen levels of IVF offspring born in Fresh ET group were significantly higher than those of Frozen ET group and NC group at all time points in the early pregnancy. There was no significant difference in the levels of T4 and TSH between Fresh ET group and NC group except that FT4 levels were intermediate between Fresh ET group and NC group. The levels of T4, FT4 and estrogen on the day of hCG were positively correlated. Conclusion: Pregnant women with ovulation induction therapy were in a hyperestrogenic state beyond the physiological range in the early pregnancy, which is the study of intrauterine hyperestrogen. Preferred exposure models. Over-physiological estradiol levels during embryonic development increase the risk of thyroid dysfunction in IVF offspring. Frozen embryo transfer reduces the risk of thyroid disease in offspring by avoiding intrauterine estrogen-rich environments. Part II Thyroid function in offspring of mice born in intrauterine high estrogen environments Objectives: To study the growth and thyroid function of offspring mice born in hyperestrogenic uterine environment and to determine the risk of adult diseases caused by hyperestrogenic uterine environment. Thyroid gland and blood samples were collected at the 18th day of pregnancy, 3rd and 8th weeks after birth. The birth weight, 3rd and 8th week weight of the offspring of the control group (C) and the high female offspring were measured. The thyroid hormone levels of the offspring were detected by radioimmunoassay; the estrogen levels in the pregnant rats were detected by ELISA kit; the thyroid morphology was observed by HE staining; and the thyroid fluorescence quantitative analysis was performed. The expression of related genes was detected by PCR and immunohistochemistry; the methylation level of CpG island in Pax8 promoter region was detected by bisulfite sequencing; the effect of 17 beta-estradiol on thyroid follicular cell proliferation and the expression of thyroid transcription factor Pax8 and methyltransferase Dnmt 3A were studied in vitro. Results: The gestation time and litter size were different. In addition, the body weight of the offspring of the high female dose group was significantly lower than that of the control group at 0-5 weeks, and there was no significant difference between the male and the control group. The expression of Pax8 and Tpo in the offspring of the high female group was significantly higher than that of the control group at 3 weeks, but there was no significant difference at 8 weeks. In addition, estrogen can promote the proliferation of thyroid follicular cell lines in a concentration-dependent manner, up-regulate the Pax8 gene and down-regulate the Dnmt3a gene in vitro. Conclusion: During the sensitive period of fetal thyroid development in early pregnancy, high concentration of estrogen stimulates the proliferation of thyroid follicular cell lines. Exposure to hormones may alter the methylation of thyroid function-related genes, leading to abnormal thyroid function in offspring. Part III Epigenetic regulation of long-stranded noncoding RNA in thyroid cells exposed to high estrogen. Objective: To study the effects of high estrogen exposure on long-stranded noncoding of thyroid follicular cells. Objective: To screen and verify the biological functions of thyroid-associated lncRNA and explore its molecular mechanisms, to evaluate the role of lncRNA in the development of thyroid dysfunction induced by high estrogen, and to determine the long-chain non-coding RNA that can predict the occurrence of adult diseases caused by high estrogen in utero. Three cases of thyroid follicular cells in estrogen treatment group and three cases in control group were selected for the analysis of lncRNAs expression chip. Real-time quantitative PCR was used to verify the six different lncRNAs in estrogen-treated thyroid follicular cell lines. Bioinformatics was used to analyze the results of the chip, and the target lncRNA was screened out in nthy ori 3-1 cells. Real-time quantitative PCR was used to verify the changes of the expression of lncDSG1 after estrogen treatment. The expression of MDM2 and thyroid cell proliferation were observed after the expression of lncDSG1 was specifically reduced by less than interfering RNA. Results: 188 lncRNAs differentially expressed in thyroid follicular cells exposed to high female were screened and up-regulated by lncRNAs 111. Six notably different lncRNAs were identified in nthy ori 3-1 cells, which were consistent with the results of the chip. The CNC network diagram suggested that LNC DSG1 might affect thyroid function. Further cell experiments confirmed that estrogen could up-regulate the expression of LNC DSG1 and MDM2 genes, but up-regulate the expression of LNC DSG1 and MDM2 genes. Conclusion: High estrogen exposure can induce the alteration of the expression profile of lncRNAs. Estrogen up-regulates the expression of lncRNADSG1 and promotes the expression of MDM2. Interference with lncRNA DSG1 can down-regulate the expression of MDM2 and inhibit the proliferation of thyroid follicular cells. RNA DSG1 may be involved in epigenetic regulation of abnormal thyroid cell function induced by high estrogen exposure.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714.8

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