宫外生长迟缓对肺血管影响及传代效应的表观遗传机制研究

发布时间：2018-06-02 15:51

  本文选题：宫外生长迟缓 + 肺血管内皮　； 参考：《浙江大学》2015年博士论文

【摘要】：背景及目的： 生命早期是个体发育的关键窗口期(critical window),关系到整个生命周期的发育及健康,甚至可影响到子孙后代。自上世纪末Barker假说的提出后,大量流行病学以及实验室研究已经证实在这一时期营养环境的改变是导致个体成年期心血管代谢性疾病发病风险增加的诱因,并且这种效应可影响到子一代,子二代甚至一直遗传下去。 官外生长迟缓(Extrauterine growth restriction, EUGR)指婴儿生后的生长发育各计量指标(身长、体重等)在相应宫内生长速率期望值的第10百分位以下(≤生长曲线的第10百分位)。根据我国目前的出生率和早产发生率计算数据显示,每年出生约150万早产儿,其中EUGR发生率极高。最新的流行病学及动物模型研究显示EUGR可致成年期体循环及肺循环的血管功能发生改变,如发生体循环收缩压增高,肺动脉压增高,提示成年期心肺血管疾病的发生可能源于生命早期的营养打击,再一次印证了“多哈理论”即“健康与疾病的发育起源”’(development origin of Health and disease, DOHaD)学说。 表观遗传学指基因的核苷酸序列不发生改变的调控基因表达的可遗传修饰。大量动物模型研究证实表观调控机制在发育起源的成人疾病,尤其是心血管代谢性疾病中发挥重要作用。在发育可塑期,不良环境使机体DNA甲基化、组蛋白修饰发生变化,改变了一些重要基因的表达,造成细胞、器官功能失调,发育受限等不良效应而最终发生疾病。这一过程也称为发育编程(development programming)。一方面,表观遗传机制如DNA甲基化具有稳定性。越来越多研究发现,这些表观修饰的改变在生命早期不良打击时已经发生,并且能够稳定持续至成年期。另一方面,表观遗传修饰本身能够遗传的特点,使得发育起源的成人疾病或生命早期的编程效应能够遗传至子代,并且已经在各种传代动物模型研究中得到证实。 本课题组前期研究发现,EUGR大鼠在成年期发生肺动脉压增高,肺血管发生重塑。同时在血管功能调节的重要器官——内皮细胞中发现多个重要基因发生不同程度的表观遗传修饰改变,这与成年期发生肺血管功能改变密切相关。但EUGR是否在暴露后早期就已经引起表观修饰改变,影响到肺血管发育或功能；并且EUGR对父代肺血管的编程效应是否能够遗传到子代,影响子代的发育及肺血管功能。这些问题尚不清楚。 基于现有资料,我们提出假设： 1.宫外生长迟缓在暴露后已经引起肺血管内皮细胞中的重要基因发生表观遗传修饰改变,影响到肺血管功能或结构,这种修饰可持续到成年期,影响成年期功能。 2.宫外生长迟缓对父代肺血管功能影响可遗传至子代,并且由表观遗传机制介导。 第一部分 宫外生长迟缓对生后早期肺血管密度影响及表观遗传机制研究 目的： 大量流行病学研究及动物模型发现生命早期营养不良可引起成年期高血压、肺动脉高压等疾病。这些心肺血管功能的失调可以追溯到生命早期不良环境暴露初期,已经引起机体重要基因的表达改变,影响到血管功能或结构,如血管密度稀疏。而表观遗传机制如DNA甲基化及组蛋白修饰在其中发挥了重要作用,并且这些修饰可以持续到成年期。我们的前期研究已经发现,EUGR可引起大鼠成年期肺动脉压增高,本研究旨在明确EUGR是否在大鼠生后早期就已经引起肺血管功能或结构的改变,并进一步明确可能引起这种效应的重要基因表达及相关的表观遗传调控机制。 方法： 1.模型建立：正常怀孕SD大鼠安置,将分娩后得到的新生仔鼠在生后24小时内以雌雄1：1比例随机分配到正常组Control (每笼8-10只仔鼠),EUGR组(每笼18-20只仔鼠)。母鼠随机分配到每笼喂养仔鼠,予随机饮食。仅保留雄性大鼠做后续研究。 2.肺动脉压评估：3周离乳雄性SD大鼠,经开胸插管测量右心室平均收缩压。9周成年雄性大鼠,经颈静脉置管术测量平均肺动脉压。 3.血管稀疏度评估：3周及9周大鼠肺组织免疫组化染色,以von-Willebrand factor(vWF)染色作为血管内皮标记,在病理切片中对肺血管进行计数。 4.免疫磁珠分选法提取肺血管内皮细胞,荧光定量RT-qPCR检测内皮细胞中Notch1基因及下游基因Hes-1,Hey-1,Hey-2的表达水平。 5.染色质免疫沉淀(chromatin immunoprecipitation, ChIP)分析Notch1基因启动子区组蛋白修饰情况；重亚硫酸盐测序分析Notch1基因启动子区CG位点的甲基化程度。 结果： 1.3周离乳大鼠右心室平均收缩压两组间无显著差异。9周成年期大鼠肺动脉平均压：EUGR组较Control组显著升高。3周离乳大鼠免疫组化染色显示,EUGR组肺血管密度低于Control组(P0.05)。9周成年大鼠两组肺血管密度无显著差异。 2.3周及9周EUGR肺血管内皮细胞中Notch1基因表达较Control组显著下降,且下游基因Hes-1表达也显著下降。 3.3周及9周Notch1基因近端启动子区EUGR组蛋白H3K27met3修饰水平较同龄Control显著增高。3周及9周EUGR组Notch1基因远端启动子区的CG位点较Control组发生甲基化程度增高。 结论： EUGR引起生后早期肺血管发育受限及成年期肺动脉压增高,与肺血管内皮细胞中Notch1表达下降密切相关。而Notch1基因启动子区组蛋白修饰及DNA甲基化发生改变可能是Notch1表达下降的潜在机制,并且这些修饰可持续到成年期。第二部分 宫外生长迟缓对子代成年期肺动脉压的影响及表观遗传机制研究目的： 生命早期不良环境不仅能够影响父代成年期健康,引起心血管代谢性疾病发病风险增高,而且这种不良效应还能够遗传到子代。这便是发育编程的传代效应。研究发现,DNA甲基化修饰是介导这一过程的重要分子机制。我们的前期研究已经发现EUGR可致成年期肺动脉压增高,肺血管功能失调。但这种不良效应能否遗传到子代尚不清楚。 方法： 1.传代模型建立：将成年期F0父代大鼠进行两两交配后,雌鼠分笼待产,所生的新生鼠根据其父代父母来源分为以下4组。母鼠予随机饮食。仔鼠出生3周后离乳,按性别分笼予随机饮食直至成年期。(1)对照组雄鼠+对照组雌鼠(C♂-C♀)(2)EUGR雄鼠+对照组雌鼠(E♂-C♀)(3)EUGR雄鼠+EUGR雌鼠(E♂-E♀)(4)对照组雄鼠+EUGR雌鼠(C♂-E♀) 2.体重评估：分别在出生、离乳期3周、成年期9周称重。 3.肺动脉压评估：9周成年大鼠,经颈静脉置管术测量平均肺动脉压。 4.免疫磁珠分选法提取肺血管内皮细胞,全基因组甲基化芯片检测父代及子代的DNA甲基化修饰情况,结果运用多种生物信息学方法分析。(1)差异甲基化遗传的情况(2)差异甲基化基因(启动子区)的功能分析(3)差异甲基化区域定位及motif预测 结果： 1.子代大鼠体重各组之间无显著差异。 2.9周E♂-C♀组雌性肺动脉压较C♂-C♀雌性显著下降(P0.001),雄性子代肺动脉压未见显著变化。 3.全基因组DNA甲基化修饰分析,子代中发生差异甲基化修饰区域的数量较父代显著减少；父代中与血压调控相关的基因DNA甲基化修饰发生遗传丢失；父代与子代差异甲基化修饰的共同区域预测到包含AP2结合域在内的功能motif。 结论： EUGR对父代肺血管功能的影响能够遗传给子代,表现为性别差异遗传特点的代偿表型。从全基因组水平分析两代的DNA甲基化修饰特点发现,父代中参与血压调控基因的甲基化修饰印迹的遗传丢失以及差异甲基化修饰区域联合转录因子协同调节基因转录这两方面因素可能是EUGR传代效应以及子代表现为代偿表型的潜在机制。
[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锛，

本文编号：1969333