【摘要】：研究背景:急性心肌梗死(AMI)是冠狀動(dòng)脈性疾病(CAD)急性發(fā)作的一種表現(xiàn)形式,主要是由不穩(wěn)定性動(dòng)脈粥樣硬化斑塊破裂所致。目前已知關(guān)于動(dòng)脈粥樣硬化的病理機(jī)制主要涉及細(xì)胞炎癥、氧化應(yīng)激、血小板功能等多個(gè)代謝過程。盡管前期全基因組關(guān)聯(lián)研究(GWAS)發(fā)現(xiàn)CAD的發(fā)生與遺傳變異有關(guān),但這些遺傳變異僅能解釋10%的CAD發(fā)生。目前認(rèn)為低頻率以及罕見的遺傳變異可能與冠心病的發(fā)生有關(guān)。SIRT2屬于sirtuin蛋白家族的一員,主要存在于哺乳動(dòng)物體內(nèi),是一種高度保守依賴煙堿胺腺嘌呤二核苷酸(NAD+)的第Ⅲ類組蛋白去乙�；�。SIRT2在基因組穩(wěn)定性、新陳代謝、炎癥、氧化應(yīng)激、自噬以及在調(diào)節(jié)血小板及血管內(nèi)皮功能方面發(fā)揮著重要的作用。在基因表達(dá)過程中,啟動(dòng)子可調(diào)節(jié)基因表達(dá)水平變化,因此,本研究推測(cè)SIRT2基因的遺傳變異在AMI的發(fā)病機(jī)制中可能存在重要的作用,力求通過研究SIRT2基因啟動(dòng)子區(qū)域的遺傳變異所引起的基因表達(dá)水平的變化來(lái)探討AMI的發(fā)生與SIRT2基因的關(guān)系。研究目的:首先在兩組人群中通過測(cè)序探討低頻遺傳變異以及單核苷酸多態(tài)性(SNPs)對(duì)AMI組和對(duì)照組人群發(fā)病的影響;其次通過構(gòu)建攜帶SIRT2基因野生型及遺傳變異位點(diǎn)啟動(dòng)子的pGL3-basic報(bào)告基因載體,檢測(cè)遺傳變異導(dǎo)致的基因表達(dá)水平的變化。通過對(duì)SIRT2基因啟動(dòng)子遺傳及功能變異的研究,探討SIRT2基因在AMI發(fā)病機(jī)制中的作用。研究方法:1、本研究最終納入375例新發(fā)AMI病例和377例健康對(duì)照人群,分別收集兩組人群臨床基線資料并提取全基因組DNA。2、根據(jù)NCBI基因數(shù)據(jù)庫(kù)提供的人SIRT2基因啟動(dòng)子序列設(shè)計(jì)引物,采用PCR方法擴(kuò)增SIRT2基因啟動(dòng)子目的片段,直接測(cè)序后統(tǒng)計(jì)分析SIRT2的基因序列變異。3、將測(cè)序發(fā)現(xiàn)的攜帶SIRT2基因啟動(dòng)子序列變異位點(diǎn)及野生型目的基因片段構(gòu)建到pGL3-basic報(bào)告基因載體,將構(gòu)建好的pGL3-basic報(bào)告基因載體及內(nèi)參質(zhì)粒pRL-TK通過脂質(zhì)體共轉(zhuǎn)染HEK293及H9c2細(xì)胞,通過Promega雙熒光報(bào)告基因分析儀分析SIRT2基因啟動(dòng)子的相對(duì)熒光素酶活性,檢測(cè)基因遺傳變異導(dǎo)致基因啟動(dòng)子轉(zhuǎn)錄活性的變化。研究結(jié)果:1、通過測(cè)序,共發(fā)現(xiàn)17個(gè)DNA序列變異(DSVs)位點(diǎn),包括5個(gè)SNPs位點(diǎn)。其中發(fā)現(xiàn)3個(gè)新的雜合DSVs,分別為g.38900270AG、g.38899853CT和g.38900888_91delTAAA,僅存在于3個(gè)AMI病人中,在正常對(duì)照組中尚未發(fā)現(xiàn)。在對(duì)照組中發(fā)現(xiàn)5個(gè)新的DSVs,分別為g.38900562CT,g.38900413AC,g.38900030GA,g.38899925AC 和 g.38899852CT,而在 AMI 組中未發(fā)現(xiàn)上述DSVs。其余4個(gè)新的雜合突變位點(diǎn)和5個(gè)SNPs在對(duì)照組及AMI組中均有發(fā)現(xiàn),但無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。2、成功構(gòu)建pGL3-basic報(bào)告基因載體,分別為pGL3-WT(野生型SIRT2基因啟動(dòng)子)、pGL3-38900907G、pGL3-38900888_91del、pGL3-38900562T、pGL3-38900413C、pGL3-38900291G、pGL3-38900270G、pGL3-38900030A、pGL3-38899925C、pGL3-38899903C、pGL3-38899853T、pGL3-38899852T 和pGL3-38899781G。3、通過脂質(zhì)體體外瞬時(shí)轉(zhuǎn)染HEK293及H9c2細(xì)胞,檢測(cè)攜帶不同DSVs的SIRT2基因啟動(dòng)子的相對(duì)熒光素酶表達(dá)活性。(1)在HEK293細(xì)胞中,僅在AMI組中發(fā)現(xiàn)DSVs可影響SIRT2基因啟動(dòng)子轉(zhuǎn)錄活性:g.38900888_91delTAAA(P0.05)和 g.38900270AG(P0.01)可顯著降低SIRT2基因啟動(dòng)子的轉(zhuǎn)錄活性;g.38899853CT顯著增加SIRT2基因啟動(dòng)子的轉(zhuǎn)錄活性(P0.01)。將僅在對(duì)照組中發(fā)現(xiàn)5個(gè)DSVs(g.38900562CT,g.38900413AC,g.38900030GA,g.38899925AC 和 g.38899852CT)轉(zhuǎn)染至HEK293細(xì)胞后,尚未發(fā)現(xiàn)可顯著改變SIRT2基因啟動(dòng)子的轉(zhuǎn)錄活性(P0.05)。同時(shí) 4 個(gè) DSVs[g.38900907AG(rs4803006),g.38900291CG(rs2053071),g.38899903TC和g.38899781CG]均存在于對(duì)照組及AMI組中,將其相應(yīng)的表達(dá)載體轉(zhuǎn)染至HEK293細(xì)胞后,仍未發(fā)現(xiàn)可顯著改變SIRT2基因啟動(dòng)子的轉(zhuǎn)錄活性(P0.05)。(2)將 AMI 組中發(fā)現(xiàn)的 g.38900888_91delTAAA 和 g.38900270AG 轉(zhuǎn)染至H9c2細(xì)胞中,發(fā)現(xiàn)可顯著降低SIRT2基因啟動(dòng)子的轉(zhuǎn)錄活性(P0.01);g.38899853CT轉(zhuǎn)染后可顯著增加SIRT2基因啟動(dòng)子的轉(zhuǎn)錄活性(P0.01)。在AMI組及對(duì)照組出現(xiàn)相似頻率的位點(diǎn)g.38900907AG(rs4803006)和g.38900291CG(rs2053071)轉(zhuǎn)染至H9c2細(xì)胞后,未發(fā)現(xiàn)可顯著改變SIRT2基因啟動(dòng)子的轉(zhuǎn)錄活性(P0.05)。研究結(jié)論:通過對(duì)SIRT2基因啟動(dòng)子遺傳及功能學(xué)變異的研究發(fā)現(xiàn),在AMI病例中發(fā)現(xiàn)的低頻序列變異位點(diǎn)可能會(huì)影響SIRT2基因的轉(zhuǎn)錄活性,導(dǎo)致其表達(dá)水平的變化,進(jìn)而在心肌梗死的發(fā)生發(fā)展中起著重要的作用,為心肌梗死的預(yù)防和治療提供了潛在的靶點(diǎn)。
[Abstract]:Background: acute myocardial infarction (AMI) is a manifestation of acute attack of coronary artery disease (CAD). It is mainly caused by the rupture of unstable atherosclerotic plaque. The pathological mechanism of atherosclerosis is known to be mainly involved in many metabolic processes, such as cell inflammation, oxygenation stress, platelet function, and so on. The whole genome association study (GWAS) found that the occurrence of CAD is associated with genetic variation, but these genetic variations can only explain 10% of the occurrence of CAD. Low frequency and rare genetic variation may be associated with the occurrence of coronary heart disease that.SIRT2 belongs to a member of the sirtuin family, which is mainly in mammals, and is highly conservative. The third class histone deacetylase.SIRT2 of nicotinamide adenine adenine dinucleotide (NAD+) plays an important role in genomic stability, metabolism, inflammation, oxidative stress, autophagy, and the regulation of platelet and vascular endothelial function. In the process of gene expression, promoter can regulate the change of gene expression level. Therefore, this study It is presumed that the genetic variation of SIRT2 gene may have an important role in the pathogenesis of AMI. To explore the relationship between the occurrence of AMI and the SIRT2 gene by studying the changes in the gene expression level of the genetic variation in the promoter region of the SIRT2 gene, the purpose of this study is to explore the low frequency inheritance by sequencing in two groups of people. The effect of mutation and single nucleotide polymorphism (SNPs) on the incidence of AMI and control groups; secondly, by constructing a pGL3-basic reporter vector carrying the SIRT2 gene and the promoter of the genetic variation site, the change of gene expression level caused by genetic variation was detected. The study on the genetic and functional variation of the promoter of the SIRT2 gene was carried out. The role of SIRT2 gene in the pathogenesis of AMI was investigated. 1. The study was finally included in 375 new AMI cases and 377 healthy controls. The clinical baseline data of two groups of people were collected and the whole genome DNA.2 was extracted respectively. The primers were designed according to the SIRT2 gene promoter sequence provided by the NCBI gene database, and the PCR method was used. The target fragment of SIRT2 gene promoter was amplified and sequenced to analyze the gene sequence variation.3 of SIRT2, and the sequence of mutations of the promoter sequence and the wild type target gene fragment of the SIRT2 gene were constructed to the pGL3-basic reporter gene carrier, and the constructed pGL3-basic reporter gene vector and the internal reference plasmid pRL-TK were used to pass the lipid. The plastids were co transfected with HEK293 and H9c2 cells, and the relative luciferase activity of SIRT2 promoter was analyzed by Promega double fluorescent reporter gene analyzer, and the changes of gene promoter transcriptional activity were detected by genetic variation. The results were as follows: 1, 17 DNA sequences (DSVs), including 5 SNPs loci, were found by sequencing. 3 new heterozygous DSVs, g.38900270AG, g.38899853CT and g.38900888_91delTAAA, were found only in 3 AMI patients, which were not found in the normal control group. 5 new DSVs were found in the control group, g.38900562CT, g.38900413AC, g.38900030GA, g.38899925AC and g.38899852CT in the AMI group. The remaining 4 new heterozygous mutation sites and 5 SNPs were found in the control group and the AMI group, but there was no statistical significance (P0.05).2. The pGL3-basic reporter gene carrier was successfully constructed, pGL3-WT (wild type SIRT2 gene promoter), pGL3-38900907G, pGL3-38900888_91del, pGL3-38900562T, pGL3-38900413C, pGL3-38900291G, etc. 38900030A, pGL3-38899925C, pGL3-38899903C, pGL3-38899853T, pGL3-38899852T and pGL3-38899781G.3 were transiently transfected into HEK293 and H9c2 cells in vitro by liposomes to detect the relative luciferase expression activity of the SIRT2 gene promoter with different DSVs. (1) in HEK293 cells, only in the AMI group could affect the promoter of the gene promoter. Transcriptional activity: g.38900888_91delTAAA (P0.05) and g.38900270AG (P0.01) significantly reduced the transcriptional activity of the SIRT2 gene promoter, and g.38899853CT significantly increased the transcriptional activity of the SIRT2 gene promoter (P0.01). Only 5 DSVs (g.38900562CT, g.38900413AC, g.38900030GA, P0.01) could be found in the control group. After 93 cells, there was no significant change in the transcriptional activity (P0.05) of the SIRT2 gene promoter, and 4 DSVs[g.38900907AG (rs4803006), g.38900291CG (rs2053071), g.38899903TC and g.38899781CG] all existed in the control group and the AMI group. After transfection of its corresponding expression vector to HEK293 cells, no significant changes were found in SIRT2 basis. The transcriptional activity of promoter (P0.05). (2) transfection of g.38900888_91delTAAA and g.38900270AG found in group AMI into H9c2 cells could significantly reduce the transcriptional activity of SIRT2 gene promoter (P0.01). G.38899853CT transfection could significantly increase the transcriptional activity of the SIRT2 gene promoter (P0.01). Similar to the AMI group and the control group. After transfection of frequency sites g.38900907AG (rs4803006) and g.38900291CG (rs2053071) to H9c2 cells, there was no significant change in the transcriptional activity of the SIRT2 gene promoter (P0.05). Conclusion: the study of the genetic and functional variation of the promoter of SIRT2 gene found that the low frequency sequence found in the AMI case may affect the mutation site in the AMI case. The transcriptional activity of SIRT2 gene leads to changes in its expression level and plays an important role in the occurrence and development of myocardial infarction, which provides potential targets for the prevention and treatment of myocardial infarction.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R542.22

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