本文選題：宮外生長遲緩 + 肺血管內(nèi)皮�。� 參考：《浙江大學》2015年博士論文

【摘要】：背景及目的： 生命早期是個體發(fā)育的關鍵窗口期(critical window),關系到整個生命周期的發(fā)育及健康,甚至可影響到子孫后代。自上世紀末Barker假說的提出后,大量流行病學以及實驗室研究已經(jīng)證實在這一時期營養(yǎng)環(huán)境的改變是導致個體成年期心血管代謝性疾病發(fā)病風險增加的誘因,并且這種效應可影響到子一代,子二代甚至一直遺傳下去。 官外生長遲緩(Extrauterine growth restriction, EUGR)指嬰兒生后的生長發(fā)育各計量指標(身長、體重等)在相應宮內(nèi)生長速率期望值的第10百分位以下(≤生長曲線的第10百分位)。根據(jù)我國目前的出生率和早產(chǎn)發(fā)生率計算數(shù)據(jù)顯示,每年出生約150萬早產(chǎn)兒,其中EUGR發(fā)生率極高。最新的流行病學及動物模型研究顯示EUGR可致成年期體循環(huán)及肺循環(huán)的血管功能發(fā)生改變,如發(fā)生體循環(huán)收縮壓增高,肺動脈壓增高,提示成年期心肺血管疾病的發(fā)生可能源于生命早期的營養(yǎng)打擊,再一次印證了“多哈理論”即“健康與疾病的發(fā)育起源”’(development origin of Health and disease, DOHaD)學說。 表觀遺傳學指基因的核苷酸序列不發(fā)生改變的調(diào)控基因表達的可遺傳修飾。大量動物模型研究證實表觀調(diào)控機制在發(fā)育起源的成人疾病,尤其是心血管代謝性疾病中發(fā)揮重要作用。在發(fā)育可塑期,不良環(huán)境使機體DNA甲基化、組蛋白修飾發(fā)生變化,改變了一些重要基因的表達,造成細胞、器官功能失調(diào),發(fā)育受限等不良效應而最終發(fā)生疾病。這一過程也稱為發(fā)育編程(development programming)。一方面,表觀遺傳機制如DNA甲基化具有穩(wěn)定性。越來越多研究發(fā)現(xiàn),這些表觀修飾的改變在生命早期不良打擊時已經(jīng)發(fā)生,并且能夠穩(wěn)定持續(xù)至成年期。另一方面,表觀遺傳修飾本身能夠遺傳的特點,使得發(fā)育起源的成人疾病或生命早期的編程效應能夠遺傳至子代,并且已經(jīng)在各種傳代動物模型研究中得到證實。 本課題組前期研究發(fā)現(xiàn),EUGR大鼠在成年期發(fā)生肺動脈壓增高,肺血管發(fā)生重塑。同時在血管功能調(diào)節(jié)的重要器官——內(nèi)皮細胞中發(fā)現(xiàn)多個重要基因發(fā)生不同程度的表觀遺傳修飾改變,這與成年期發(fā)生肺血管功能改變密切相關。但EUGR是否在暴露后早期就已經(jīng)引起表觀修飾改變,影響到肺血管發(fā)育或功能；并且EUGR對父代肺血管的編程效應是否能夠遺傳到子代,影響子代的發(fā)育及肺血管功能。這些問題尚不清楚。 基于現(xiàn)有資料,我們提出假設： 1.宮外生長遲緩在暴露后已經(jīng)引起肺血管內(nèi)皮細胞中的重要基因發(fā)生表觀遺傳修飾改變,影響到肺血管功能或結構,這種修飾可持續(xù)到成年期,影響成年期功能。 2.宮外生長遲緩對父代肺血管功能影響可遺傳至子代,并且由表觀遺傳機制介導。 第一部分 宮外生長遲緩對生后早期肺血管密度影響及表觀遺傳機制研究 目的： 大量流行病學研究及動物模型發(fā)現(xiàn)生命早期營養(yǎng)不良可引起成年期高血壓、肺動脈高壓等疾病。這些心肺血管功能的失調(diào)可以追溯到生命早期不良環(huán)境暴露初期,已經(jīng)引起機體重要基因的表達改變,影響到血管功能或結構,如血管密度稀疏。而表觀遺傳機制如DNA甲基化及組蛋白修飾在其中發(fā)揮了重要作用,并且這些修飾可以持續(xù)到成年期。我們的前期研究已經(jīng)發(fā)現(xiàn),EUGR可引起大鼠成年期肺動脈壓增高,本研究旨在明確EUGR是否在大鼠生后早期就已經(jīng)引起肺血管功能或結構的改變,并進一步明確可能引起這種效應的重要基因表達及相關的表觀遺傳調(diào)控機制。 方法： 1.模型建立：正常懷孕SD大鼠安置,將分娩后得到的新生仔鼠在生后24小時內(nèi)以雌雄1：1比例隨機分配到正常組Control (每籠8-10只仔鼠),EUGR組(每籠18-20只仔鼠)。母鼠隨機分配到每籠喂養(yǎng)仔鼠,予隨機飲食。僅保留雄性大鼠做后續(xù)研究。 2.肺動脈壓評估：3周離乳雄性SD大鼠,經(jīng)開胸插管測量右心室平均收縮壓。9周成年雄性大鼠,經(jīng)頸靜脈置管術測量平均肺動脈壓。 3.血管稀疏度評估：3周及9周大鼠肺組織免疫組化染色,以von-Willebrand factor(vWF)染色作為血管內(nèi)皮標記,在病理切片中對肺血管進行計數(shù)。 4.免疫磁珠分選法提取肺血管內(nèi)皮細胞,熒光定量RT-qPCR檢測內(nèi)皮細胞中Notch1基因及下游基因Hes-1,Hey-1,Hey-2的表達水平。 5.染色質(zhì)免疫沉淀(chromatin immunoprecipitation, ChIP)分析Notch1基因啟動子區(qū)組蛋白修飾情況；重亞硫酸鹽測序分析Notch1基因啟動子區(qū)CG位點的甲基化程度。 結果： 1.3周離乳大鼠右心室平均收縮壓兩組間無顯著差異。9周成年期大鼠肺動脈平均壓：EUGR組較Control組顯著升高。3周離乳大鼠免疫組化染色顯示,EUGR組肺血管密度低于Control組(P0.05)。9周成年大鼠兩組肺血管密度無顯著差異。 2.3周及9周EUGR肺血管內(nèi)皮細胞中Notch1基因表達較Control組顯著下降,且下游基因Hes-1表達也顯著下降。 3.3周及9周Notch1基因近端啟動子區(qū)EUGR組蛋白H3K27met3修飾水平較同齡Control顯著增高。3周及9周EUGR組Notch1基因遠端啟動子區(qū)的CG位點較Control組發(fā)生甲基化程度增高。 結論： EUGR引起生后早期肺血管發(fā)育受限及成年期肺動脈壓增高,與肺血管內(nèi)皮細胞中Notch1表達下降密切相關。而Notch1基因啟動子區(qū)組蛋白修飾及DNA甲基化發(fā)生改變可能是Notch1表達下降的潛在機制,并且這些修飾可持續(xù)到成年期。第二部分 宮外生長遲緩對子代成年期肺動脈壓的影響及表觀遺傳機制研究目的： 生命早期不良環(huán)境不僅能夠影響父代成年期健康,引起心血管代謝性疾病發(fā)病風險增高,而且這種不良效應還能夠遺傳到子代。這便是發(fā)育編程的傳代效應。研究發(fā)現(xiàn),DNA甲基化修飾是介導這一過程的重要分子機制。我們的前期研究已經(jīng)發(fā)現(xiàn)EUGR可致成年期肺動脈壓增高,肺血管功能失調(diào)。但這種不良效應能否遺傳到子代尚不清楚。 方法： 1.傳代模型建立：將成年期F0父代大鼠進行兩兩交配后,雌鼠分籠待產(chǎn),所生的新生鼠根據(jù)其父代父母來源分為以下4組。母鼠予隨機飲食。仔鼠出生3周后離乳,按性別分籠予隨機飲食直至成年期。(1)對照組雄鼠+對照組雌鼠(C♂-C♀)(2)EUGR雄鼠+對照組雌鼠(E♂-C♀)(3)EUGR雄鼠+EUGR雌鼠(E♂-E♀)(4)對照組雄鼠+EUGR雌鼠(C♂-E♀) 2.體重評估：分別在出生、離乳期3周、成年期9周稱重。 3.肺動脈壓評估：9周成年大鼠,經(jīng)頸靜脈置管術測量平均肺動脈壓。 4.免疫磁珠分選法提取肺血管內(nèi)皮細胞,全基因組甲基化芯片檢測父代及子代的DNA甲基化修飾情況,結果運用多種生物信息學方法分析。(1)差異甲基化遺傳的情況(2)差異甲基化基因(啟動子區(qū))的功能分析(3)差異甲基化區(qū)域定位及motif預測 結果： 1.子代大鼠體重各組之間無顯著差異。 2.9周E♂-C♀組雌性肺動脈壓較C♂-C♀雌性顯著下降(P0.001),雄性子代肺動脈壓未見顯著變化。 3.全基因組DNA甲基化修飾分析,子代中發(fā)生差異甲基化修飾區(qū)域的數(shù)量較父代顯著減少；父代中與血壓調(diào)控相關的基因DNA甲基化修飾發(fā)生遺傳丟失；父代與子代差異甲基化修飾的共同區(qū)域預測到包含AP2結合域在內(nèi)的功能motif。 結論： EUGR對父代肺血管功能的影響能夠遺傳給子代,表現(xiàn)為性別差異遺傳特點的代償表型。從全基因組水平分析兩代的DNA甲基化修飾特點發(fā)現(xiàn),父代中參與血壓調(diào)控基因的甲基化修飾印跡的遺傳丟失以及差異甲基化修飾區(qū)域聯(lián)合轉錄因子協(xié)同調(diào)節(jié)基因轉錄這兩方面因素可能是EUGR傳代效應以及子代表現(xiàn)為代償表型的潛在機制。
[Abstract]:Background and purpose:
Early life is the key window period of individual development (critical window), related to the development and health of the whole life cycle, and even to future generations. After the introduction of the Barker hypothesis at the end of the last century, a large number of epidemiological and laboratory studies have confirmed that the changes in the nutritional environment in this period have led to individual adult life. The risk of increased risk of metabolic diseases may be affected by the effect of the metabolic syndrome, and the offspring may even be inherited from the two generation.
Extrauterine growth restriction (EUGR) means less than tenth percentile of each measurement index (body length, weight, etc.) in the corresponding intrauterine growth rate (less than tenth percentile of the growth curve) after birth. According to the current rate of birth and the incidence of preterm birth in China, the birth rate is about 150 per year. The prevalence of EUGR is very high. The latest epidemiological and animal model studies have shown that EUGR can cause changes in the vascular function of the adult body circulation and pulmonary circulation, such as the increase of systemic circulation systolic pressure and the increase of pulmonary arterial pressure. It confirms the "Doha theory", that is, the theory of development origin of Health and disease (DOHaD).
Epigenetics refers to the genetic modification of the gene expression that does not change the nucleotide sequence of the gene. A large number of animal models have proved that the apparent regulatory mechanism plays an important role in the development of adult disease, especially in cardiovascular metabolic diseases. In the developmental plasticity period, the adverse environment makes the body DNA methylation, histone modification Changes have changed the expression of some important genes, resulting in adverse effects such as cell, organ dysfunction, development restriction and eventual disease. This process is also called developmental programming (development programming). On the one hand, epigenetic mechanisms such as DNA methylation have stability. More and more studies have found that these apparent modifications are modified. On the other hand, epigenetic modification, on the other hand, can inherit the genetic characteristics of the epigenetic modification itself, allowing the programming effects of the adult disease or early life to be inherited to the offspring, and has been confirmed in the study of various generation animal models.
In the previous study, we found that the pulmonary artery pressure increased in adult EUGR rats and the pulmonary vascular remodeling. At the same time, different degrees of epigenetic modification were found in the important organs of vascular function regulating endothelial cells, which were closely related to the changes of pulmonary vascular function in adult stage, but EUGR was the same. In the early days of exposure, apparent modification has been caused to affect pulmonary vascular development or function; and whether the programming effect of EUGR on the parent lung vessels can be inherited to the offspring, affects the development of the offspring and the function of the pulmonary vessels. These problems are not yet clear.
Based on the existing data, we put forward the hypothesis:
1. extrauterine growth retardation has caused epigenetic modification of the important genes in the pulmonary vascular endothelial cells after exposure, affecting the function or structure of the pulmonary vessels, which can continue to adulthood and affect adult function.
2. the effect of extrauterine growth retardation on parent pulmonary vascular function can be inherited to the offspring and is mediated by epigenetic mechanism.
Part one
Effects of extrauterine growth retardation on pulmonary vascular density and epigenetic mechanism in early postnatal stage
Objective:
A large number of epidemiological studies and animal models have found that early malnutrition in life can cause diseases of adult hypertension and pulmonary hypertension. These disorders can be traced to the early stage of adverse environmental exposure, which have caused changes in the expression of important genes in the body, affecting vascular function or structure, such as blood vessel density. The epigenetic mechanism, such as DNA methylation and histone modification, plays an important role, and these modifications can last to adulthood. Our previous studies have found that EUGR can cause increased pulmonary arterial pressure in adult rats. This study aims to determine whether EUGR has caused pulmonary vascular function in the early post birth of rats. Or structural changes, and further clarify the important gene expression and related epigenetic regulatory mechanisms that may cause this effect.
Method錛，

本文編號：1969333