子痫前期对子代HPA轴的影响及其调控机制研究

发布时间：2018-04-25 16:57

  本文选题：子痫前期 + 母鼠　； 参考：《浙江大学》2014年博士论文

【摘要】：成人代谢性疾病是危害人类健康的主要疾病之一,近年来其发病率呈逐年上升趋势。“成年疾病的胎儿起源学说(fetal origin of adult disease,FOAD)'’提出成人期心血管疾病、2型糖尿病、代谢综合症,肿瘤等多种慢性疾病发病源于胎儿期宫内发育不良。子痫前期(preeclampsia, PE)是妊娠期特有的常见疾病,以高血压和蛋白尿为主要临床特征,是引起孕产妇和围产儿发病和死亡的一个重要原因。越来越多的流行病学调查提示,PE子代发生成年期血压、糖代谢异常的风险显著增加。 官内营养不良或不平衡时,处于发育敏感期胎儿组织器官在结构和功能上将会发生永久性或程序性改变,各种激素轴重新设置,大脑等重要器官的发育得到一定的保障,同时必然导致其它器官的营养供给减少,这些变化增加了出生后对各种慢性病的易感性。糖皮质激素(Glucocorticoid,GC)在妊娠期间对母体和胎儿都起着极其重要的作用,大量的研究发现,当胎儿处于官内缺氧、酸中毒、胎儿窘迫及胎盘损伤等病理状态下,可使胎儿暴露于过量的糖皮质激素中,导致胎儿出生体重减低和子代下丘脑一垂体一肾上腺轴(HPA轴)重塑,是导致成年疾病发生的关键因素。11β-羟基类固醇脱氢酶II型(11β-hydroxysteroid dehydrogenase type2,11β-HSD2)是HPA轴在糖皮质激素作用调节中的关键。多项研究证实11β-HSD2与高血压、代谢综合症等疾病发生密切相关。11β-HSD2能将有活性的GC代谢为无活性的代谢产物来调节母体和胎儿体内的GC平衡,以保证胎儿发育所需要的适宜GC环境。子痫前期对子代代谢性疾病的发生及机制的研究甚少,子痫前期所致的不良官内环境是否会引起胎儿HPA轴功能的改变?是否影响出生后子代幼年乃至成年的健康?此过程的可能的调控机制又是什么?这是本研究拟阐明的几个关键环节。 本研究首先建立子病前期动物模型,观察子代发育及血压、代谢相关指标的改变,探讨子痫前期子代代谢性疾病发生发展过程,研究HPA轴的变化及11β-HSD2的调控改变；其次,我们分析子痫前期胎儿脐带血糖皮质激素水平及11β-HSD2基因启动子的甲基化水平改变,探索其表观遗传学调控机制；观察子痫前期患者的胎盘组织中11β-HSD2的表达及启动子甲基化的改变；最后通过贝莉发育量表对人类子痫前期子代的健康状况进行随访和流行病学调查,观察子代血压及神经系统发育的改变。本研究以子代高血压作为切入点,揭示母体-胎盘-子代可能存在以11β-HSD2介导的HPA轴程序性变化,通过研究子痫前期与子代高血压发生发展之间的关系,探讨成人疾病的胎儿源性机制,为临床干预提供科学的依据。 第一部分子痫前期动物模型的建立及其子代HPA轴改变的机制研究 目的： 建立子病前期大鼠动物模型,观察子痫前期对成年期子代大鼠血压的影响,探讨观察子鼠下丘脑-垂体。肾上腺轴相应器官及激素的病理生理变化及其与11β-HSD2的相关性,探索子痫前期的子代大鼠胎源性疾病的可能机制。 方法： 通过一氧化氮合酶抑制剂-L-NAME在妊娠期的干预来建立子痫前期的动物模型。观察子鼠脑、肾脏组织及海马神经元的病理改变,酶联免疫吸附测定法测定子鼠促肾上腺皮质激素(ACTH)、皮质酮浓度变化；免疫组化及RT-PCR检测肾脏11β-HSD2水平；免疫组化检测肾脏组织和海马组织11β-HSD2、GR水平；免疫荧光检测下丘脑CRH蛋白表达。 结果： 模型组妊娠大鼠在注射L-NAME后血压、蛋白尿逐渐持续增加,与子痫前期临床表现一致,建模成功。子痫前期模型组：1.胎鼠体重显著降低(P0.001),子鼠出生后12、14、16、18及20周子鼠收缩压与对照组无显著差异(P均(0.05),出生后第20周模型组舒张压显著高于对照组(P(0.05)；2.子鼠的血浆皮质酮、ACTH水平显著高于正常对照组；3.子鼠海马神经元在电镜下有凋亡现象；4.对PE子代肾组织的免疫组化及PCR检测结果显示,11β-HSD2蛋白的表达明显低于正常妊娠子代；5.免疫荧光检测下丘脑CRH蛋白表达模型组与对照组相比表达明显降低。 结论： 子痫前期子鼠成年后血压风险增加且发生HPA轴相应器官的病理损伤及激素改变,提示子痫前期子代高糖皮质激素暴露及HPA轴的改变与子代成年高血压发生有关,并与11β-HSD2的表达调控有关。 第二部分子痫前期子代皮质醇水平及11p-HSD2 启动子甲基化变化的研究目的： 本研究拟探讨子痫前期子代脐血皮质醇水平及11p-HSD2启动子甲基化的变化及与子代代谢综合征风险的关系。 方法： 选择43例子痫前期患者(轻度25例,重度18例),运用电化学发光法测定脐血血浆皮质醇及ACTH浓度,运用MassARRAY定量分析系统测定脐血11p-HSD2启动子CpG岛甲基化水平,采用多元线性回归和线性混合模型统计分析。 结果： PE组平均血浆皮质醇水平明显高于对照组(PE,264.39±167.10nmol/L;对照组148.34±48.49nmol/L,P0.001),PE组平均血浆ACTH水平明显高于对照组(PE,26.55±18.03pmol/L；对照组14.35±11.03pmol/L,P0.01). 测得6个CpG甲基化水平数据有效。PE组HSD9-2.HSD9-3.HSD23-2、HSD23-3及平均水平明显低于对照组(0.10±0.019vs0.09±0.018:0.41±0.048vs0.39±0.056;0.17±0.096vs0.13±0.031;0.15±0.064vs0.11±0.048；0.16±0.051vs0.13±0.029),PE与11β-HSD2启动子甲基化水平正相关(r=0.325,P0.001)。 结论： PE子代处于高皮质醇、ACTH环境,可能与子代代谢性疾病发生有相关性；PE降低了子代11β-HSD2启动子甲基化水平,PE与甲基化水平存在正相关。11β-HSD2启动子甲基化的改变可能与子代代谢性疾病发病机制之一。 第三部分子痫前期患者胎盘中11β-HSD2基因表达及其甲基化水平的研究 目的： 检测子痫前期胎盘中11β-HSD2的表达及其启动子甲基化水平,明确11β-HSD2基因的甲基化水平及其和PE胎盘中11β-HSD2基因表达之间的关系。 方法： 采用免疫组织化学染色对胎盘组织病理学特征进行研究,用RT-PCR和Western blotting检测11β-HSD2mRNA和蛋白水平。用亚硫酸氢钠测序法检测11β-HSD2启动子甲基化水平。 结果： 免疫组化结果显示11β-HSD2在PE患者呈不规则地分布且其免疫反应明显减弱,11β-HSD2mRNA和蛋白水平PE组明显低于正常对照组。11β-HSD2甲基化水平在PE胎盘为(two fragments,0.6%vs.0%)而正常组为(1%vs.0.6%),两组间的甲基化水平无显著差异(P0.05)。 结论： PE孕妇胎盘11β-HSD2表达降低与子痫前期调控11β-HSD2表达的机制失调有关,推测与子代糖皮质激素的升高有关；胎盘11β-HSD2基因的表达调控机制复杂,可能不完全受DNA甲基化的影响。 第四部分子痫前期子代贝莉发育量表初步评估 目的： 对纠正胎龄为2-5.9月的子痫前期患者子代进行贝莉发育量表(BSID-Ⅱ)评估,明确子痫前期患者子代早期神经系统发育状况及血压变化,探讨可能的发病机制。 方法： 选择78例纠正胎龄2-5.9个月的子痫前期患者子代和60例同龄正常妊娠子代作为对照,进行BSID-Ⅱ发育量表测定分析及血压监测。 结果： 两组子代血压无明显差异,但PE组子代智力发展指数(MDI)、精细运动发展指数(PDI)均明显低于对照组(MDI),PE组子代出生体重≤1500g,1500-1999g与≥2500g及2000-2499g组之间,MDI和PDI存在显著差异性。 结论： PE子代在儿童期存在神经系统的发育迟缓,可能与PE子代宫内发育迟缓和早产低体重儿有关。
[Abstract]:Adult metabolic diseases are one of the major diseases that harm human health. In recent years, the incidence of the disease is increasing year by year. "Fetal origin of adult disease, FOAD", "the adult cardiovascular disease, type 2 diabetes, metabolic syndrome, tumor and other chronic diseases are derived from the intrauterine intrauterine disease. Preeclampsia (PE) is a common common disease in pregnancy. Hypertension and proteinuria are the main clinical features, and it is an important cause of the onset and death of pregnant and parturients and perinatal infants. More and more epidemiological investigations suggest that PE offspring generate annual blood pressure, and the risk of abnormal glucose metabolism is significantly increased.
In the case of malnutrition or imbalances, the structure and function of fetal tissues and organs in the developmental sensitive period will have permanent or procedural changes, the resetting of various hormone axes, the development of the important organs such as the brain are guaranteed, and the nutritional supply of other organs will be reduced, and these changes increase the postnatal effect. The susceptibility to various chronic diseases. Glucocorticoid (GC) plays an extremely important role in both the mother and the fetus during pregnancy. A large number of studies have found that the fetus is exposed to excessive glucocorticoid and the fetus is born when the fetus is in the pathological state of anoxia, acidosis, fetal distress and placental injury. The reduction of body weight and the remolding of the one adrenal axis (HPA axis) of the hypothalamus, the key factor leading to the occurrence of adult disease,.11 beta hydroxy steroid dehydrogenase II (11 beta -hydroxysteroid dehydrogenase type2,11 beta -HSD2) is the key to the regulation of the action of the HPA axis in glucocorticoid. A number of studies have confirmed that 11 beta -HSD2 and hypertension and metabolic syndrome have been confirmed. The closely related.11 beta -HSD2 can metabolize active GC into inactive metabolites to regulate the GC balance between the mother and the fetus in order to ensure the appropriate GC environment for the development of the fetus. There are few studies on the occurrence and mechanism of sub generation metabolic diseases in preeclampsia, and the adverse environment in the preeclampsia Will the fetal HPA axis function change? Does it affect the health of the young and adult offspring of the postnatal offspring? What are the possible regulatory mechanisms for this process? This is a key link in this study.
In this study, we first set up the prepredist animal model, observe the development of the progeny and blood pressure, and change the metabolic related indexes, explore the development process of the subgeneration subgeneration metabolic diseases, study the changes of HPA axis and the regulation of 11 beta -HSD2. Secondly, we analyze the level of corticosteroids in fetal umbilical cord and the 11 beta -HSD2 gene in preeclampsia fetal umbilical cord. The changes in the methylation level of the promoter and the epigenetic regulation mechanism were explored. The expression of 11 beta -HSD2 and the change of promoter methylation in placental tissues of preeclampsia patients were observed. Finally, the Bailey development scale was used to follow up the health status of the preeclampsia and to investigate the blood pressure and nervous system in the subgeneration of the preeclampsia. In this study, this study took the subgeneration hypertension as a breakthrough point and revealed that the parent placenta progeny may have the HPA axis programmed changes mediated by 11 beta -HSD2. Through the study of the relationship between preeclampsia and the occurrence and development of subgeneration hypertension, the fetal pathogenesis of adult disease is explored to provide a scientific basis for clinical intervention.
Establishment of the first animal model of preeclampsia and the mechanism of HPA axis changes in offspring
Objective:
To establish the rat model of preeclampsia, observe the effect of preeclampsia on the blood pressure of the adult rat, observe the pathological changes of the hypothalamus pituitary, the corresponding organs and hormones of the adrenal axis and the correlation with the 11 beta -HSD2, and explore the possible mechanism of the fetal disease in the preeclampsia.
Method锛，

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