本文選題：甾醇氧-乙酰轉(zhuǎn)移酶　切入點：前列腺素內(nèi)過氧化物合酶2　出處：《南通大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的Soat1基因編碼的乙酰輔酶A：膽固醇乙酰轉(zhuǎn)移酶在維持細胞內(nèi)膽固醇的動態(tài)平衡方面具有重要作用,直接或間接地影響了APP的分泌過程及Aβ的產(chǎn)生；Ptgs2基因編碼的COX能將花生四烯酸代謝成各種前列腺素類,其中前列腺素E參與了膽固醇的合成和運輸,Ptgs2過度表達可增強Aβ的作用強度,加強興奮毒性引起的神經(jīng)退行性病變。由此可推測,Ptgs2基因與Soat1基因可能在細胞內(nèi)膽固醇的運輸和轉(zhuǎn)化上存在一定的聯(lián)系,進而共同參與阿爾茨海默病(Alzheimer's disease, AD)的發(fā)病。為研究Soat1基因和Ptsg2基因在AD發(fā)病過程中協(xié)同作用的分子機制,本實驗應(yīng)用基因芯片技術(shù)、基因表達數(shù)量性狀基因座定位(expression Qutative Trati Loci, eQTL)的方法和遺傳相關(guān)度分析,借助BXD重組近交系(Recombinant Inbred, RI)小鼠,構(gòu)建Soat1和Ptgs2基因的表達調(diào)控網(wǎng)絡(luò),從全基因組水平上研究這兩個基因參與AD發(fā)病的分子機制。 方法本實驗采用BXDRI小鼠,運用eQTL定位的方法對Soat1基因(Probe set1417697_at)及Ptgs2基因(Probe set1417263_at)的表達變異及表達調(diào)控進行研究,研究兩基因在BXDRI小鼠及親本(C57BL/6J和DBA/2)海馬組織中的表達差異及差異的變化趨勢。采用區(qū)間作圖在全基因組水平分別定位控制兩基因表達變化的基因座(e-QTL)。通過檢索數(shù)據(jù)庫獲取B6和D2兩品系間的單核苷酸多態(tài)性(Single Nucleotide Polymorphisms, SNP)資料,并分析SNP對eQTL定位的影響。利用ClusterMap在線分析工具檢索BXDRI小鼠海馬組織基因組中分別受Soat1和Ptgs2基因反式調(diào)節(jié)的基因,即尋找其下游候選基因。通過皮爾森相關(guān)系數(shù)法篩選出具有高度遺傳相關(guān)性的基因,并構(gòu)建基因表達遺傳調(diào)控網(wǎng)絡(luò)。 結(jié)果RT-PCR結(jié)果顯示Soat1基因的表達水平在B6和D2兩親本的海馬組織中存在顯著差異,在B6中的表達量明顯高于D2,在BXDRI小鼠海馬組織中的平均表達量為7.959(7),表達量在各品系小鼠中呈線性變化趨勢,該結(jié)果顯示Soat1基因具備QTL定位的條件。通過全基因組區(qū)間作圖法定位Soat1基因的上游調(diào)控位點,發(fā)現(xiàn)其自身所在的基因組位置上存在一個連鎖值較高的eQTL, LRS值高達89.2,且相對應(yīng)的探針靶序列上的SNP對eQTL檢測無影響,初步判斷該基因為順式調(diào)控基因候選基因。運用cluster map在線分析工具檢索BXD重組近交系小鼠海馬組織基因組中受Soat1反式調(diào)節(jié)的基因,Soat1基因被定位到反式eQTL富集區(qū),21個基因的反式eQTL被定位到此處,排除連鎖不平衡對下游基因定位的影響,結(jié)果顯示其中的7個基因與Soat1呈顯著性偏相關(guān),由此推測這7個基因為Soat1基因的下游候選調(diào)控基因。通過皮爾森相關(guān)系數(shù)法分析Soat1基因與BXD RI小鼠海馬組織中其他基因的遺傳相關(guān)度,獲得遺傳相關(guān)度較高的(0.43)前18個基因,Ptgs2包含在其中,遺傳相關(guān)度高達0.61。 Ptgs2基因在BXD重組近交系小鼠海馬組織中平均表達量高達8.32,在親本B6小鼠海馬組織中表達為8.54,D2中表達量較低為7.86,兩親本間表達差異達1.6倍,差異具有統(tǒng)計學(xué)意義,且在BXD重組近交系各個品系間的表達變化呈線性趨勢,符合數(shù)量性狀的特征�；虮磉_數(shù)量性狀基因座定位分析發(fā)現(xiàn)控制該基因的eQTL為順式eQTL,即調(diào)控該基因表達變化的為該基因自身。該區(qū)域同時還是反式eQTL的富集區(qū),偏相關(guān)系數(shù)分析發(fā)現(xiàn)其中有8個基因為Ptgs2基因的下游調(diào)控候選基因。皮爾森相關(guān)系數(shù)分析發(fā)現(xiàn)BXD重組近交系小鼠基因組中有151個基因與Ptgs2高度相關(guān),其中前13個基因與Ptgs2的遺傳相關(guān)度到達0.59以上,Soat1基因包含在其中。 在構(gòu)建的Ptgs2與Soat1基因遺傳網(wǎng)絡(luò)中,有10個基因同時與Soat1基因和Ptgs2基因呈現(xiàn)高度相關(guān)性。其中,Dnm3、 Glul兩個基因均參與了AD的發(fā)病,Dnm3做為Dnm家族成員之一,還參與了膽固醇的運輸與代謝。由此推測,Soat1基因可能通過Dnm及Glul基因與Ptgs2基因聯(lián)系起來共同參與了AD的發(fā)病,進一步說明Soat1基因和Ptgs2協(xié)同參與了AD的發(fā)病。 結(jié)論本實驗可初步判斷Soat1、Ptgs2基因為順式調(diào)控基因,Rab2,Taz等7個基因為Soat1基因的下游候選調(diào)控基因,Vcam1、Mlstd1等8個基因為Ptgs2基因的下游候選調(diào)控基因。無論在Soat1基因遺傳相關(guān)基因網(wǎng)絡(luò)中還是在Ptgs2基因遺傳相關(guān)基因網(wǎng)絡(luò)中,Ptgs2與Soat1基因都呈現(xiàn)高度相關(guān)性。在構(gòu)建的Ptgs2與Soat1基因網(wǎng)絡(luò)中,Dnm和Glu1基因同時與Soat1和Ptgs2呈現(xiàn)高度的相關(guān)性,并參與了APP的加工和Aβ的產(chǎn)生,由此推測,Soat1基因可能通過Dnm及Glu1基因與Ptgs2基因聯(lián)系起來共同參與了AD的發(fā)病,由此進一步證明Ptgs2與Soat1基因協(xié)同參與了AD的發(fā)病過程。
[Abstract]:Objective: the Soat1 gene encoding acetyl coenzyme A: cholesterol acyltransferase plays an important role in maintaining the dynamic balance of intracellular cholesterol, directly or indirectly affect the secretion of APP and A beta; Ptgs2 gene encoding COX can be four arachidonic acid metabolism into various prostanoids, including prostaglandin E in synthesis and transport of cholesterol, overexpression of Ptgs2 can enhance the strength of A beta, neurodegeneration caused by excitotoxicity. Strengthen it can be concluded that Ptgs2 gene and Soat1 gene may be linked cholesterol transport and transformation in cells, and involved in Alzheimer's disease (Alzheimer's, disease, AD) incidence for study of the molecular mechanism of Soat1 gene and Ptsg2 gene synergistically in the pathogenesis of AD, the application of gene chip technology, gene expression quantity of gene. (expression Qutative Trati Loci base location, eQTL) correlation analysis method and genetic, using BXD recombinant inbred lines (Recombinant, Inbred, RI) mice, construct the expression regulation networks of Soat1 and Ptgs2 genes, the molecular mechanism of these two genes from the whole genome level in the pathogenesis of AD.
In this experiment we use BXDRI mice, using the method of eQTL localization of Soat1 gene (Probe set1417697_at) and Ptgs2 (Probe set1417263_at) gene expression variation and expression regulation of two genes in BXDRI mice and their parents (C57BL/6J and DBA/2) expression differences and changes in hippocampus. The interval mapping in the whole genome level were two loci positioning control gene expression changes (e-QTL). By searching the database to obtain the polymorphism of B6 and D2 between the two lines (the Single Nucleotide Polymorphisms, SNP), and analyze the effect of SNP on the eQTL location. Soat1 and Ptgs2 analysis tools to retrieve genes transactivated by gene respectively. BXDRI in the hippocampus in the mouse genome using ClusterMap online, searching for its downstream gene. Through the Pearson correlation coefficient was screened with high genetic correlation Sex gene and gene expression genetic regulation network.
Results RT-PCR results showed that the expression level of Soat1 gene had significant differences in B6 and D2 of two parents in the hippocampus, the expression in B6 was significantly higher than that of D2, the average expression in the hippocampus of BXDRI mice was 7.959 (7), the expression of linear change trend in different strains of mice, the results display with QTL localization of Soat1 gene. The condition of the upstream control sites of whole genome interval mapping method to locate the Soat1 gene, it is found that there is a higher eQTL of its own value chain the genomic position, LRS value is as high as 89.2, and the corresponding probe target sequence of SNP had no effect on the detection of eQTL, preliminary judgment this gene is a candidate gene CIS gene regulation. Using cluster map online analysis tools to retrieve BXD recombinant inbred mice hippocampus genome by Soat1 transregulated genes, the Soat1 gene was mapped to the trans eQTL enrichment zone, 21 trans eQTL gene was mapped to the exclusion of linkage disequilibrium effects on downstream gene localization, the results showed that there was a significant correlation of 7 genes and Soat1, which indicated that the 7 genes downstream of Soat1 gene as candidate gene. Through Pearson correlation coefficient method for analysis of genetic correlation of other Soat1 gene BXD and RI in hippocampus of mice in the inheritance of acquired high correlation (0.43) before the 18 genes, Ptgs2 included, genetic correlation as high as 0.61.
Inbred Ptgs2 gene in recombinant BXD strain mice in the hippocampus of the average level of up to 8.32, in the hippocampus of B6 mice in parents was 8.54. The expression of D2 gene expression is low 7.86, two differential expression between the parents was 1.6 times, the difference was statistically significant, and the BXD expression of recombinant inbred lines of various products between a linear trend, in line with the characteristics of quantitative traits. The gene expression of QTL mapping analysis showed that the control of the gene eQTL for CIS eQTL, which regulate the gene expression changes of the gene itself. The region is also the enrichment of trans eQTL, partial correlation analysis shows that there are 8 as a downstream gene candidate gene Ptgs2. Pearson correlation analysis showed that BXD recombinant inbred strain mouse genome with 151 genes highly correlated with Ptgs2, which is the first 13 Ptgs2 gene and genetic correlation to reach 0.59 On the other hand, the Soat1 gene is included in it.
In the Ptgs2 and Soat1 gene genetic network construction, there are 10 genes and Soat1 gene and Ptgs2 gene were highly correlated. Among them, Dnm3 and Glul two genes involved in the pathogenesis of AD, Dnm3 as a member of the Dnm family, is involved in the transport and metabolism of cholesterol. By this hypothesis, Soat1 gene may be linked by Dnm and Glul genes and Ptgs2 genes involved in the pathogenesis of AD, suggested that Soat1 gene and Ptgs2 co participate in the pathogenesis of AD.
Conclusion this experiment can determine the initial Soat1, Ptgs2 gene cis regulatory genes, Rab2, Taz and other 7 genes downstream of Soat1 gene candidate genes, Vcam1, Mlstd1 and other 8 genes as candidate gene Ptgs2 downstream regulated genes. Both genes involved in Soat1 gene genetic network in network or in the Ptgs2 gene genetic correlation gene, Ptgs2 gene and Soat1 gene are highly correlated. In Ptgs2 and Soat1 network construction in Dnm, and the Glu1 gene with Soat1 and Ptgs2 showed a high degree of correlation, and participated in the processing of the APP and A beta, it is speculated that Soat1 gene may be linked by Dnm and Glu1 gene and Ptgs2 gene participate in the pathogenesis of AD, which further proved that Ptgs2 and Soat1 genes involved in the pathogenesis of AD.

【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R749.16

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相關(guān)期刊論文 前1條

1 趙發(fā)國,王蔭華,陳彪,楊靜芳,馬秋蘭,湯哲,劉宏軍,董秀敏,馮秀麗;乙酰輔酶A:膽固醇乙酰轉(zhuǎn)移酶基因單核苷酸多態(tài)性與散發(fā)性阿爾茨海默病的相關(guān)性研究[J];中國神經(jīng)免疫學(xué)和神經(jīng)病學(xué)雜志;2005年05期

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本文編號：1565203