脂肪酸调控TET1介导血管内皮细胞去甲基化的代间效应及机制研究

Being exposed to maternal malnutrition during pregnancy increases the long-term risk of hypertension in offspring. Abnormal lipid metabolism during pregnancy can induce chronic diseases of offspring after birth. However, the underlying mechanism is still unclear. In the clinical follow-up study we found that high maternal triglyceride levels in the third trimester increased the risk for adult hypertension in offspring, and obtained a group of differentially methylated genes related to cardiovascular function by whole genome high-throughput methylation analysis of offspring’s cord blood. Here, we plan to further study the intergenerational transmission effect and mechanism of fatty acid on TET1 mediated demethylation of vascular endothelial cells. We will collect umbilical endothelial cells, cord blood of newborns, and peripheral blood of children by clinical follow-up, detecting methylation levels of candidate genes by pyrosequencing technique. We are going to use the established high-fat-diet pregnant rat model, detecting the expression and methylation alteration of TET1 and candidate genes of blood vessels in offspring, and exploring the intergenerational transmission effect when exposure to high maternal triglyceride. By using HUVEC cells in culture, we try to identify the role of fatty acid on the process of demethylation via TET1 knockdown followed by fatty acid treatment, investigate the molecular mechanism of fatty acid-mediated FOXO3a’s transcriptional regulation of TET1 by ChIP and luciferase assay. With these studies above, we want to provide new ideas for the pathogenesis study of fetal original hypertension induced by in utero exposure to over-nutrition and provide a theoretical basis for the prevention and control of chronic disease.

宫内营养不良增加子代远期高血压风险，母亲孕期脂代谢异常可引发子代出生后的慢性疾病但机制不详。我们临床随访发现母亲孕晚期高甘油三酯水平增加子代成年高血压发生风险，通过高通量筛查子代脐血全基因组甲基化，获得一组与心血管功能相关的差异基因。本项目拟在此基础上深入探讨脂肪酸调控TET1介导的血管内皮细胞去甲基化及代间传递效应。通过临床随访收集新生儿脐带血管内皮细胞及脐血和儿童外周血，利用焦磷酸盐测序技术检测靶基因的甲基化水平；利用已建立的孕期高甘油三酯大鼠模型，获取子代血管组织检测甲基化水平、TET1及靶基因改变，探讨高脂暴露的代间传递效应；通过HUVEC细胞培养，敲减TET1后进行脂肪酸处理明确脂肪酸在干扰甲基化中的作用，利用ChIP及荧光素酶报告系统分析脂肪酸介导FOXO3a对TET1转录调控的分子机制。为宫内营养过剩干扰胎儿源性高血压发病机理研究开拓新思路和慢性疾病的源头防控提供新理论依据。

胎儿宫内发育是生命起始的最初阶段，其正常的发育关系到个体生命的成长和健康。对生命发育早期状况，特别是宫内发育环境与成年期慢性疾病之间关系的探索已成为现代医学的研究热点。本研究聚焦母亲孕期宫内营养过剩尤其是高脂暴露与子代远期心血管代谢性疾病风险的相关性，通过流行病学调查和队列研究及前瞻性跟踪随访，发现母亲孕期高甘油三酯水平可增加子代出生体重和早产的发生，且子代在儿童期即出现心血管代谢风险因子水平的升高以及成年后高血压的发生风险显著增加。尽管已有潜在的证据支持胎儿宫内高脂暴露与远期疾病的相关性，但是其中的机制仍不清楚。因此，我们通过高通量筛查子代脐血全基因组甲基化，获得一组与脂代谢紊乱和心血管功能相关的差异基因。利用焦磷酸盐测序进一步验证关键差异基因的甲基化改变。此外，通过建立孕期高脂喂养的动物模型，证实了孕期高脂暴露可致子代成年后血压持久的升高。深入的机制探究发现高脂暴露子代呈现持续升高的血清leptin水平和脂肪组织和淋巴细leptin基因持续显著的高表达和低甲基化状态，同时伴有去甲基化酶TET1表达的升高。通过体外分离间充质干细胞，诱导成脂分化过程中加不同浓度的脂肪酸，结果发现高浓度的脂肪酸可增加leptin基因的表达和促进去甲基化酶TET1的高表达，导致leptin基因的低甲基化。本项目从临床问题引发基础研究，通过对甲基化芯片进行深入分析，筛查出发生甲基化改变的心血管相关目标基因，并作进一步功能验证和机制研究；通过建立妊娠期高血脂水平的大鼠模型，从表观遗传学的层面阐明宫内高脂肪酸暴露致特定基因重编程引发子代心血管风险的可能机制，为实现远期慢性疾病的源头控制提供理论依据。随着本课题的开展，为与之相关的一些研究如其他代谢性疾病的宫内源性及其表观遗传机制等开拓新思路，奠定理论基础，并为阻断疾病发生，提供新的线索。