發(fā)布時(shí)間：2018-04-10 22:39

  本文選題：DNA甲基化 + 甲基化芯片　； 參考：《南京大學(xué)》2015年碩士論文

【摘要】：冠心病(coronary artery disease, CAD):具有多基因遺傳性,其發(fā)生過(guò)程往往是多因素、多疾病協(xié)同的結(jié)果,存在眾多危險(xiǎn)因素,被認(rèn)為是受表觀遺傳學(xué)規(guī)律控制的人類重要疾病。DNA甲基化是最早發(fā)現(xiàn)的表觀遺傳修飾方式之一,也是目前該領(lǐng)域研究的熱點(diǎn)。甲基化修飾雖不改變DNA序列,但對(duì)基因的表達(dá)與沉默起重要的調(diào)節(jié)作用。急性冠脈綜合征(acute coronary syndrome, ACS)包括不穩(wěn)定型心絞痛(unstable angina, UA)、急性心肌梗死(acute myocardial infarction, AMI)等各種急性心肌缺血引起的臨床綜合征,發(fā)病突然,致死率高,是造成猝死的重要原因。DNA甲基化在ACS的發(fā)生發(fā)展過(guò)程中扮演著怎樣的角色,以及在病理生理機(jī)制中起到什么樣的作用有待進(jìn)一步的探索與研究。目的：(1)應(yīng)用高密度基因芯片(450K Infinium Methylation BeadChip)技術(shù)分析ACS患者與正常人外周血全基因組DNA甲基化差異水平,尋找ACS患者DNA甲基化水平顯著變化的基因,并進(jìn)行功能富集分析以探索其在ACS疾病發(fā)生、發(fā)展過(guò)程中所發(fā)揮的作用。(2)應(yīng)用甲基化特異性PCR(methylation-specific PCR, MS-PCR/MSP)檢測(cè)CAD患者與正常對(duì)照組中C型1類尼曼-匹克蛋白(Niemann-Pick C1 protein, NPC1)基因啟動(dòng)子區(qū)甲基化水平的差異,為冠心病早期診斷、治療提供的新靶標(biāo)和實(shí)驗(yàn)依據(jù)。方法：(1)收集ACS患者和正常人(作為對(duì)照)全血標(biāo)本各3份,分別提取DNA并進(jìn)行亞硫酸鹽轉(zhuǎn)化,與Illumina HD 450K Infinium Methylation BeadChip芯片雜交,對(duì)450,000多個(gè)甲基化位點(diǎn)進(jìn)行檢測(cè),覆蓋96%的CpG島；針對(duì)Illumine 450K甲基化芯片中的探針甲基化位點(diǎn),進(jìn)行數(shù)據(jù)標(biāo)準(zhǔn)化(Control normalization)預(yù)處理,計(jì)算探針?biāo)降募谆潭鹊乃?Beta score);對(duì)高甲基化基因進(jìn)行GO功能分析(Gene Ontology Analysis, GO)和京都基因與基因組百科全書(shū)(Kyoto encyclopedia of Genes and Genomes, KEGG)功能分析。(2)收集64例正常人、37例穩(wěn)定型冠心病(stable coronary artery disease, SCAD)患者和55例ACS患者的全血標(biāo)本,分別提取DNA并進(jìn)行亞硫酸鹽轉(zhuǎn)化,用MS-PCR法檢測(cè)NPC1啟動(dòng)子區(qū)甲基化水平,分析患者與正常人的甲基化水平差異。結(jié)果：(1)ACS患者與正常人全血基因組間共發(fā)現(xiàn)770個(gè)基因存在DNA甲基化水平的差異(Beta score0.2, p0.05),其中甲基化程度降低的基因有640個(gè),升高的基因有130個(gè)。GO和KEGG功能分析結(jié)果顯示甲基化差異的基因功能主要集中在細(xì)胞信號(hào)通路、糖脂代謝、細(xì)胞粘附分子和細(xì)胞周期細(xì)胞凋亡,這些基因的DNA甲基化水平升高可能是導(dǎo)致ACS的原因之一。(2)SCAD患者全血DNA的NPC1啟動(dòng)子區(qū)甲基化水平明顯高于正常對(duì)照組和ACS患者,而ACS患者NPC1啟動(dòng)子區(qū)甲基化水平較正常對(duì)照組無(wú)明顯差異。結(jié)論：(1)ACS患者全基因組甲基化水平較正常對(duì)照組發(fā)生顯著變化,這可能是導(dǎo)致ACS發(fā)生、發(fā)展的重要因素之一。(2)冠心病患者NPC1甲基化水平明顯升高,可能與AS的斑塊形成和AS逐漸嚴(yán)重時(shí)斑塊穩(wěn)定相關(guān)。對(duì)這些基因的甲基化水平進(jìn)行檢測(cè)將有益于冠心病,特別是ACS的早期診斷和病情監(jiān)控。
[Abstract]:Coronary heart disease (coronary artery disease, CAD): with polygenic inheritance, the process is often multi factors, multi disease collaborative results, there are many risk factors, was found to be affected by the apparent human diseases.DNA methylation genetics control law is the most early one of epigenetic modification, is currently the focus in this field. Methylation does not alter the sequence of DNA, but the expression of gene silencing and plays an important regulatory role. Acute coronary syndrome (acute coronary, syndrome, ACS) including unstable angina (unstable, angina, UA), acute myocardial infarction (acute myocardial infarction, AMI) by a variety of acute myocardial ischemia in clinical syndrome, sudden onset, high fatality rate, is the important cause of sudden death caused by.DNA methylation plays a role in what happened in the process of ACS development, as well as the pathologist To further explore and study what kind of role play mechanism. Objective: (1) the application of high density gene chip (450K Infinium Methylation BeadChip) technology, different levels of DNA methylation in peripheral blood of ACS patients and normal people, looking for ACS DNA in patients with significant changes in methylation level and gene. The function enrichment analysis to explore its occurrence in ACS disease, which play a role in the process of development. (2) by methylation specific PCR (methylation-specific, PCR, MS-PCR/MSP) detection of CAD patients and normal control group C type 1 - Niemann PEAK (Niemann-Pick C1 protein, NPC1 protein) gene promoter hypermethylation differences the level of the early diagnosis of coronary heart disease, and provide experimental basis for new target treatment. Methods: (1) collected from ACS patients and normal people (as control) whole blood specimens were collected from 3, DNA were extracted and sulfite Salt transformation, hybridization and Illumina HD 450K Infinium Methylation BeadChip chip was used to detect the methylation sites of the more than 450000, covering 96% of the CpG island methylation sites for the probe; Illumine 450K methylation chip, data standardization (Control normalization) pretreatment, the methylation level computing probe level level (Beta score GO); functional analysis of high methylation gene (Gene Ontology Analysis, GO) and Kyoto Encyclopedia of genes and genomes (Kyoto Encyclopedia of Genes and Genomes, KEGG) functional analysis. (2) collected from 64 normal subjects, 37 patients with stable coronary heart disease (stable coronary artery disease, SCAD) and 55 patients with whole blood samples ACS patients, DNA were extracted and the sulfite conversion, detection of NPC1 promoter methylation level by MS-PCR method, analysis of methylation level in patients with normal differences. Results: (1) ACS patients and normal human blood genome were found between the 770 gene methylation level of DNA (Beta score0.2 P0.05), the decrease of methylation of 640 genes, 130 genes were increased.GO and KEGG function analysis showed that the gene methylation difference in the main function focus on cell signaling pathways, lipid metabolism, cell adhesion molecules, cell cycle and apoptosis, increased the level of DNA methylation of these genes may be the cause of ACS. (2) SCAD patients blood DNA NPC1 promoter methylation was significantly higher than the normal control group and ACS patients, and ACS patients NPC1 methylation levels compared with normal control group, no significant difference. Conclusion: (1) genomic DNA methylation levels in ACS patients compared with normal control group changed significantly, which may lead to the occurrence of ACS, the development of one of the important factors. (2) coronary heart The level of NPC1 methylation is significantly higher in patients with disease. It may be related to plaque formation in AS and plaque in AS gradually. Detection of methylation level of these genes will be beneficial for early diagnosis and monitoring of coronary heart disease, especially ACS.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R541.4

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