本文選題：肥胖 + DNA甲基化��； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：第一章葉酸補(bǔ)充對高脂飲食誘導(dǎo)的胰島素抵抗的影響及其機(jī)制研究背景:肥胖癥的流行是引起胰島素抵抗、高血壓、2型糖尿病和心血管疾病等慢性疾病發(fā)生率增加的主要原因。肥胖相關(guān)的胰島素抵抗是遺傳和環(huán)境因素共同作用的結(jié)果。這些因素在疾病發(fā)展期間通過表觀遺傳修飾發(fā)生相互作用,表觀遺傳學(xué)標(biāo)志可能解釋了生活方式和疾病風(fēng)險(xiǎn)之間的聯(lián)系。白色脂肪組織(WAT)在肥胖相關(guān)的胰島素抵抗中起著重要作用。然而,WAT的功能異常以及在促進(jìn)肥胖相關(guān)胰島素抵抗中的確切機(jī)制尚不清楚。葉酸(FA)通過提供一碳單位在S-腺苷甲硫氨酸的合成通路中發(fā)揮重要作用,FA補(bǔ)充能夠改變?nèi)蚪MDNA的甲基化特點(diǎn),預(yù)防DNA損傷,促進(jìn)DNA修復(fù)。已有研究發(fā)現(xiàn),在伴有2型糖尿病的肥胖癥患者,FA補(bǔ)充改善了胰島素抵抗、降低了血漿同型半胱氨酸(Hcy)水平,同時(shí)改善了血糖控制。然而,對于FA補(bǔ)充在肥胖風(fēng)險(xiǎn)和胰島素抵抗中的作用仍然缺乏系統(tǒng)性研究。我們因此擬采用全基因組甲基化測序的方法,直接探索FA補(bǔ)充對小鼠脂肪組織DNA甲基化特點(diǎn)的影響,并檢測這些作用帶來的功能改變。方法:雄性C57BL/6J小鼠分別給予高脂飲食(HFD),正常飲食(ND),或HFD補(bǔ)充FA飲食(FAD),喂養(yǎng)10周后,獲取小鼠的附睪脂肪組織,利用甲基化免疫共沉淀測序(MeDIP-seq)技術(shù),對小鼠脂肪組織進(jìn)行全基因組甲基化測序。通過亞硫酸氫鹽測序的方法驗(yàn)證高通量的測序結(jié)果,隨后,采用熒光定量PCR(RT-qPCR)方法檢測相關(guān)甲基化改變基因的相應(yīng)表達(dá)量變化。結(jié)果:小鼠經(jīng)HFD喂養(yǎng)10周后,相比對照組,體重、附睪脂肪重量均顯著增加,同時(shí)HFD組小鼠空腹血糖和血清胰島素水平也明顯升高。FA補(bǔ)充顯著減少了 HFD誘導(dǎo)的脂肪累積,降低了空腹血糖水平,改善了胰島素抵抗。MeDIP-seq結(jié)果顯示,差異甲基化區(qū)(DMRs)分布在整個(gè)小鼠基因組上,在HFD組小鼠,出現(xiàn)明顯的DNA高甲基化。在FAD組與HFD組之間,共鑒定出3787個(gè)差異甲基化注釋基因,通路分析這些差異甲基化基因(DMGs)發(fā)現(xiàn),與insulinsecretion,pancreatic secretion,type2 diabetes信號(hào)通路密切相關(guān)。亞硫酸氫鹽測序驗(yàn)證了這些通路中的基因(Adcy3和Rapgef4)甲基化改變,基因表達(dá)量改變同樣證實(shí),相比HFD組,FA補(bǔ)充引起Adcy3和Rapgef4基因的表達(dá)顯著增加。結(jié)論:FA補(bǔ)充飲食改善了 HFD誘導(dǎo)的胰島素抵抗,減少了脂肪塊累積,誘導(dǎo)了與能量代謝和胰島素分泌相關(guān)基因的甲基化和表達(dá)量改變。第二章葉酸補(bǔ)充對肥胖相關(guān)心功能異常和心肌纖維化的作用及其機(jī)制研究背景:肥胖被認(rèn)為是不合理的脂肪組織累積和功能異常引起的能量平衡異常,導(dǎo)致了2型糖尿病和心血管疾病的盛行。大量研究證實(shí),肥胖引起了心肌結(jié)構(gòu)重塑和心功能異常。然而,這些改變的潛在機(jī)制尚不完全清楚。氧化應(yīng)激和炎癥被認(rèn)為參與了多種類型的心血管疾病的發(fā)病機(jī)制和疾病進(jìn)展。有研究發(fā)現(xiàn),氧化應(yīng)激在肥胖相關(guān)的心血管異常中起著關(guān)鍵作用。在糖尿病心臟病患者,使用抗氧化劑能夠明顯緩解心肌纖維化和炎癥反應(yīng)。葉酸作為一種抗氧化劑,近來發(fā)現(xiàn)能夠減少心肌缺血期間的活性氧產(chǎn)物的產(chǎn)生,緩解心功能異常。一項(xiàng)對隨機(jī)對照實(shí)驗(yàn)的meta分析也證實(shí),葉酸補(bǔ)充能夠明顯降低心血管疾病的風(fēng)險(xiǎn)。FA的抗氧化作用被認(rèn)為是預(yù)防心血管疾病的關(guān)鍵因素。此外,高Hcy血癥也被發(fā)現(xiàn)與高風(fēng)險(xiǎn)心血管疾病相關(guān),飲食中補(bǔ)充FA則可以將Hcy轉(zhuǎn)換為蛋氨酸降低血漿Hcy水平。因此,FA被認(rèn)為具有預(yù)防ROS產(chǎn)生和緩解高Hcy血癥的作用,而ROS和Hcy與心功能異常密切相關(guān)。然而,FA在肥胖相關(guān)性心肌病中的作用尚未有研究報(bào)道。方法:雄性6周齡C57BL/6J小鼠分別給予ND,HFD和FAD,喂養(yǎng)14周后,在麻醉?xiàng)l件下,經(jīng)胸壁超聲心動(dòng)圖檢測各組小鼠的心臟功能。同時(shí)測量小鼠體重變化、心臟重量、血糖水平、心臟組織中FA和Hcy含量,以及心肌氧化應(yīng)激狀態(tài),組織病理觀察各組小鼠心肌的病理變化。結(jié)果:HFD引起了小鼠空腹血糖升高,體重和心臟重量增加。FA補(bǔ)充增加了心肌組織中的FA含量,改善了 HFD誘導(dǎo)的空腹血糖水平,降低了小鼠心臟重量和HFD誘導(dǎo)的心臟Hcy含量升高。此外,相比ND組,HFD組小鼠的心臟射血分?jǐn)?shù)和短軸縮短率均明顯下降,而FA補(bǔ)充顯著改善了 HFD誘導(dǎo)的小鼠心功能異常。同時(shí),FA補(bǔ)充預(yù)防了 HFD引起的心肌纖維化。相比對照組,HFD明顯增加了心臟組織MDA含量,降低了抗氧化酶GSH和CAT活性,而FA補(bǔ)充顯著改善了 HFD誘導(dǎo)的心肌氧化應(yīng)激狀態(tài)。結(jié)論:FA補(bǔ)充顯著降低了肥胖小鼠的空腹血糖水平,減少了心臟Hcy含量,逆轉(zhuǎn)了肥胖相關(guān)的心功能異常。這些FA補(bǔ)充對心臟功能的改善可能通過緩解氧化應(yīng)激和減少心肌纖維化介導(dǎo)。
[Abstract]:Chapter 1 the effect of folic acid supplementation on insulin resistance induced by high fat diet and its mechanism research background: obesity is the main cause of the increase in the incidence of chronic diseases such as insulin resistance, hypertension, type 2 diabetes and cardiovascular disease. Obesity related insulin resistance is the result of the combination of genetic and environmental factors. These factors interact through epigenetic modification during the development of the disease. Epigenetic markers may explain the link between lifestyle and the risk of disease. White fat tissue (WAT) plays an important role in obesity related insulin resistance. However, the dysfunction of WAT and the promotion of obesity related insulin can be used. The exact mechanism of resistance is not clear. Folic acid (FA) plays an important role in the synthesis pathway of S- adenosine methionine by providing a single carbon unit, FA supplementation can change the methylation of whole genome DNA, prevent DNA damage and promote DNA repair. The study found that FA supplementation improved the islets in obese patients with type 2 diabetes. Hormone resistance reduces plasma homocysteine (Hcy) levels and improves blood glucose control. However, there is still a lack of systematic study on the role of FA supplementation in obesity and insulin resistance. We should therefore use a complete genome sequencing method to explore the DNA methylation characteristics of FA supplementation in mouse adipose tissue directly. Method: male C57BL/6J mice were given high fat diet (HFD), normal diet (ND), or HFD supplemented FA diet (FAD). After feeding 10 weeks, the epididymal adipose tissue of mice was obtained, and the methylation immunoprecipitation sequencing (MeDIP-seq) technique was used to carry out the whole genome methylation of the mouse adipose tissue. The results of high throughput sequencing were verified by the method of hydrogen sulfite sequencing. Subsequently, the corresponding expression changes of related methylation altered genes were detected by fluorescence quantitative PCR (RT-qPCR). Results: after 10 weeks of feeding in HFD, the weight and epididymal fat weight of mice increased significantly compared with the control group, while the fasting blood glucose in the group HFD mice was at the same time. The level of.FA supplementation significantly reduced the accumulation of fat induced by HFD, reduced the level of fasting blood glucose, and improved the results of insulin resistance.MeDIP-seq, and the difference methylation area (DMRs) was distributed throughout the genome of the mice, and in the HFD group, the obvious hypermethylation of DNA was found. Between the FAD group and the HFD group, a total of DNA hypermethylation was found. 3787 differential methylation annotation genes were identified. Pathway analysis of these differentially methylated genes (DMGs) was found to be closely related to insulinsecretion, pancreatic secretion, and type2 diabetes signaling pathways. Compared with the HFD group, FA supplementation caused a significant increase in the expression of Adcy3 and Rapgef4 genes. Conclusion: FA supplementation improves the insulin resistance induced by HFD, reduces the accumulation of fat blocks, induces the methylation and expression of genes related to energy metabolism and insulin secretion. The second chapter folic acid supplementation to obesity is concerned with abnormal function and myocardium. The role of fibrosis and its mechanism research background: obesity is considered to be an abnormal energy balance caused by the accumulation and dysfunction of adipose tissue, which leads to the prevalence of type 2 diabetes and cardiovascular disease. A large number of studies have confirmed that obesity has caused myocardial remodeling and cardiac dysfunction. However, the potential mechanisms of these changes are not yet completed. It is fully clear that oxidative stress and inflammation are considered to be involved in the pathogenesis and disease progression of various types of cardiovascular diseases. Studies have found that oxidative stress plays a key role in obesity related cardiovascular abnormalities. In patients with diabetes, the use of antioxidants can significantly alleviate myocardial fibrosis and inflammatory reactions. As an antioxidant, it has recently been found to reduce the production of reactive oxygen products during myocardial ischemia and relieve cardiac dysfunction. A meta analysis of randomized controlled trials also confirmed that folic acid supplementation can significantly reduce the risk of cardiovascular disease. The antioxidant effect of.FA is considered to be a key factor in preventing cardiovascular disease. In addition, high H CY has also been found to be associated with high risk cardiovascular disease. Dietary supplementation of FA can convert Hcy to methionine to reduce plasma Hcy levels. Therefore, FA is considered to have the effect of preventing ROS production and alleviating hyperlipidemia, and ROS and Hcy are closely related to cardiac dysfunction. However, the role of FA in obesity related cardiomyopathy has not yet been studied. Methods: the male C57BL/6J mice of 6 weeks of age were given ND, HFD and FAD respectively. After 14 weeks of feeding, the cardiac function of each group was detected by the chest wall echocardiography. The body weight change, heart weight, blood glucose level, the content of FA and Hcy in the heart tissue, and the oxidative stress state of the heart were measured, and the pathological observation of the myocardium was observed. Pathological changes in myocardium of mice in each group. Results: HFD caused the increase of fasting blood glucose in mice. The increase of weight and heart weight.FA supplemented the FA content in the myocardium, improved the HFD induced fasting blood glucose level, reduced the weight of the heart of the mice and the Hcy content in the heart induced by HFD. In addition, compared to the ND group, the cardiac ejection of the HFD mice. FA supplementation significantly improved cardiac dysfunction induced by HFD, and FA supplementation prevented myocardial fibrosis induced by HFD. HFD significantly increased the MDA content of cardiac tissue, reduced the activity of antioxidant enzyme GSH and CAT, and FA supplementation significantly improved HFD induced myocardial oxygen. Conclusion: FA supplementation significantly reduces fasting blood glucose levels in obese mice, reduces cardiac Hcy content and reverses obesity related cardiac dysfunction. These FA supplements may be mediated by alleviating oxidative stress and reducing myocardial fibrosis.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R589.2

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