本文選題：子癇前期 + 母鼠�。� 參考：《浙江大學(xué)》2014年博士論文

【摘要】：成人代謝性疾病是危害人類健康的主要疾病之一,近年來其發(fā)病率呈逐年上升趨勢�！俺赡昙膊〉奶浩鹪磳W(xué)說(fetal origin of adult disease,FOAD)'’提出成人期心血管疾病、2型糖尿病、代謝綜合癥,腫瘤等多種慢性疾病發(fā)病源于胎兒期宮內(nèi)發(fā)育不良。子癇前期(preeclampsia, PE)是妊娠期特有的常見疾病,以高血壓和蛋白尿為主要臨床特征,是引起孕產(chǎn)婦和圍產(chǎn)兒發(fā)病和死亡的一個重要原因。越來越多的流行病學(xué)調(diào)查提示,PE子代發(fā)生成年期血壓、糖代謝異常的風(fēng)險顯著增加。 官內(nèi)營養(yǎng)不良或不平衡時,處于發(fā)育敏感期胎兒組織器官在結(jié)構(gòu)和功能上將會發(fā)生永久性或程序性改變,各種激素軸重新設(shè)置,大腦等重要器官的發(fā)育得到一定的保障,同時必然導(dǎo)致其它器官的營養(yǎng)供給減少,這些變化增加了出生后對各種慢性病的易感性。糖皮質(zhì)激素(Glucocorticoid,GC)在妊娠期間對母體和胎兒都起著極其重要的作用,大量的研究發(fā)現(xiàn),當(dāng)胎兒處于官內(nèi)缺氧、酸中毒、胎兒窘迫及胎盤損傷等病理狀態(tài)下,可使胎兒暴露于過量的糖皮質(zhì)激素中,導(dǎo)致胎兒出生體重減低和子代下丘腦一垂體一腎上腺軸(HPA軸)重塑,是導(dǎo)致成年疾病發(fā)生的關(guān)鍵因素。11β-羥基類固醇脫氫酶II型(11β-hydroxysteroid dehydrogenase type2,11β-HSD2)是HPA軸在糖皮質(zhì)激素作用調(diào)節(jié)中的關(guān)鍵。多項研究證實11β-HSD2與高血壓、代謝綜合癥等疾病發(fā)生密切相關(guān)。11β-HSD2能將有活性的GC代謝為無活性的代謝產(chǎn)物來調(diào)節(jié)母體和胎兒體內(nèi)的GC平衡,以保證胎兒發(fā)育所需要的適宜GC環(huán)境。子癇前期對子代代謝性疾病的發(fā)生及機(jī)制的研究甚少,子癇前期所致的不良官內(nèi)環(huán)境是否會引起胎兒HPA軸功能的改變?是否影響出生后子代幼年乃至成年的健康?此過程的可能的調(diào)控機(jī)制又是什么?這是本研究擬闡明的幾個關(guān)鍵環(huán)節(jié)。 本研究首先建立子病前期動物模型,觀察子代發(fā)育及血壓、代謝相關(guān)指標(biāo)的改變,探討子癇前期子代代謝性疾病發(fā)生發(fā)展過程,研究HPA軸的變化及11β-HSD2的調(diào)控改變；其次,我們分析子癇前期胎兒臍帶血糖皮質(zhì)激素水平及11β-HSD2基因啟動子的甲基化水平改變,探索其表觀遺傳學(xué)調(diào)控機(jī)制；觀察子癇前期患者的胎盤組織中11β-HSD2的表達(dá)及啟動子甲基化的改變；最后通過貝莉發(fā)育量表對人類子癇前期子代的健康狀況進(jìn)行隨訪和流行病學(xué)調(diào)查,觀察子代血壓及神經(jīng)系統(tǒng)發(fā)育的改變。本研究以子代高血壓作為切入點,揭示母體-胎盤-子代可能存在以11β-HSD2介導(dǎo)的HPA軸程序性變化,通過研究子癇前期與子代高血壓發(fā)生發(fā)展之間的關(guān)系,探討成人疾病的胎兒源性機(jī)制,為臨床干預(yù)提供科學(xué)的依據(jù)。 第一部分子癇前期動物模型的建立及其子代HPA軸改變的機(jī)制研究 目的： 建立子病前期大鼠動物模型,觀察子癇前期對成年期子代大鼠血壓的影響,探討觀察子鼠下丘腦-垂體。腎上腺軸相應(yīng)器官及激素的病理生理變化及其與11β-HSD2的相關(guān)性,探索子癇前期的子代大鼠胎源性疾病的可能機(jī)制。 方法： 通過一氧化氮合酶抑制劑-L-NAME在妊娠期的干預(yù)來建立子癇前期的動物模型。觀察子鼠腦、腎臟組織及海馬神經(jīng)元的病理改變,酶聯(lián)免疫吸附測定法測定子鼠促腎上腺皮質(zhì)激素(ACTH)、皮質(zhì)酮濃度變化；免疫組化及RT-PCR檢測腎臟11β-HSD2水平；免疫組化檢測腎臟組織和海馬組織11β-HSD2、GR水平；免疫熒光檢測下丘腦CRH蛋白表達(dá)。 結(jié)果： 模型組妊娠大鼠在注射L-NAME后血壓、蛋白尿逐漸持續(xù)增加,與子癇前期臨床表現(xiàn)一致,建模成功。子癇前期模型組：1.胎鼠體重顯著降低(P0.001),子鼠出生后12、14、16、18及20周子鼠收縮壓與對照組無顯著差異(P均(0.05),出生后第20周模型組舒張壓顯著高于對照組(P(0.05)；2.子鼠的血漿皮質(zhì)酮、ACTH水平顯著高于正常對照組；3.子鼠海馬神經(jīng)元在電鏡下有凋亡現(xiàn)象；4.對PE子代腎組織的免疫組化及PCR檢測結(jié)果顯示,11β-HSD2蛋白的表達(dá)明顯低于正常妊娠子代；5.免疫熒光檢測下丘腦CRH蛋白表達(dá)模型組與對照組相比表達(dá)明顯降低。 結(jié)論： 子癇前期子鼠成年后血壓風(fēng)險增加且發(fā)生HPA軸相應(yīng)器官的病理損傷及激素改變,提示子癇前期子代高糖皮質(zhì)激素暴露及HPA軸的改變與子代成年高血壓發(fā)生有關(guān),并與11β-HSD2的表達(dá)調(diào)控有關(guān)。 第二部分子癇前期子代皮質(zhì)醇水平及11p-HSD2 啟動子甲基化變化的研究目的： 本研究擬探討子癇前期子代臍血皮質(zhì)醇水平及11p-HSD2啟動子甲基化的變化及與子代代謝綜合征風(fēng)險的關(guān)系。 方法： 選擇43例子癇前期患者(輕度25例,重度18例),運(yùn)用電化學(xué)發(fā)光法測定臍血血漿皮質(zhì)醇及ACTH濃度,運(yùn)用MassARRAY定量分析系統(tǒng)測定臍血11p-HSD2啟動子CpG島甲基化水平,采用多元線性回歸和線性混合模型統(tǒng)計分析。 結(jié)果： PE組平均血漿皮質(zhì)醇水平明顯高于對照組(PE,264.39±167.10nmol/L;對照組148.34±48.49nmol/L,P0.001),PE組平均血漿ACTH水平明顯高于對照組(PE,26.55±18.03pmol/L；對照組14.35±11.03pmol/L,P0.01). 測得6個CpG甲基化水平數(shù)據(jù)有效。PE組HSD9-2.HSD9-3.HSD23-2、HSD23-3及平均水平明顯低于對照組(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與11β-HSD2啟動子甲基化水平正相關(guān)(r=0.325,P0.001)。 結(jié)論： PE子代處于高皮質(zhì)醇、ACTH環(huán)境,可能與子代代謝性疾病發(fā)生有相關(guān)性；PE降低了子代11β-HSD2啟動子甲基化水平,PE與甲基化水平存在正相關(guān)。11β-HSD2啟動子甲基化的改變可能與子代代謝性疾病發(fā)病機(jī)制之一。 第三部分子癇前期患者胎盤中11β-HSD2基因表達(dá)及其甲基化水平的研究 目的： 檢測子癇前期胎盤中11β-HSD2的表達(dá)及其啟動子甲基化水平,明確11β-HSD2基因的甲基化水平及其和PE胎盤中11β-HSD2基因表達(dá)之間的關(guān)系。 方法： 采用免疫組織化學(xué)染色對胎盤組織病理學(xué)特征進(jìn)行研究,用RT-PCR和Western blotting檢測11β-HSD2mRNA和蛋白水平。用亞硫酸氫鈉測序法檢測11β-HSD2啟動子甲基化水平。 結(jié)果： 免疫組化結(jié)果顯示11β-HSD2在PE患者呈不規(guī)則地分布且其免疫反應(yīng)明顯減弱,11β-HSD2mRNA和蛋白水平PE組明顯低于正常對照組。11β-HSD2甲基化水平在PE胎盤為(two fragments,0.6%vs.0%)而正常組為(1%vs.0.6%),兩組間的甲基化水平無顯著差異(P0.05)。 結(jié)論： PE孕婦胎盤11β-HSD2表達(dá)降低與子癇前期調(diào)控11β-HSD2表達(dá)的機(jī)制失調(diào)有關(guān),推測與子代糖皮質(zhì)激素的升高有關(guān)；胎盤11β-HSD2基因的表達(dá)調(diào)控機(jī)制復(fù)雜,可能不完全受DNA甲基化的影響。 第四部分子癇前期子代貝莉發(fā)育量表初步評估 目的： 對糾正胎齡為2-5.9月的子癇前期患者子代進(jìn)行貝莉發(fā)育量表(BSID-Ⅱ)評估,明確子癇前期患者子代早期神經(jīng)系統(tǒng)發(fā)育狀況及血壓變化,探討可能的發(fā)病機(jī)制。 方法： 選擇78例糾正胎齡2-5.9個月的子癇前期患者子代和60例同齡正常妊娠子代作為對照,進(jìn)行BSID-Ⅱ發(fā)育量表測定分析及血壓監(jiān)測。 結(jié)果： 兩組子代血壓無明顯差異,但PE組子代智力發(fā)展指數(shù)(MDI)、精細(xì)運(yùn)動發(fā)展指數(shù)(PDI)均明顯低于對照組(MDI),PE組子代出生體重≤1500g,1500-1999g與≥2500g及2000-2499g組之間,MDI和PDI存在顯著差異性。 結(jié)論： PE子代在兒童期存在神經(jīng)系統(tǒng)的發(fā)育遲緩,可能與PE子代宮內(nèi)發(fā)育遲緩和早產(chǎn)低體重兒有關(guān)。
[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錛，

本文編號：1802180