本文選題：先天性心臟病 + 圓錐動(dòng)脈干畸形　； 參考：《復(fù)旦大學(xué)》2014年碩士論文

【摘要】：先天性心臟病(簡(jiǎn)稱先心病)是胎兒期心臟及大血管發(fā)育異常所致的先天性畸形,發(fā)病率在活產(chǎn)嬰兒中占6%0-10%0。我國(guó)每年約出生15萬先心病患兒,造成社會(huì)家庭嚴(yán)重的精神經(jīng)濟(jì)負(fù)擔(dān)。青紫型先心病是先心病中最嚴(yán)重的類型,畸形復(fù)雜,可致頑固性低氧血癥,未經(jīng)治療,早期死亡率高達(dá)80%。圓錐動(dòng)脈干畸形占青紫型先心的60%-70%。法洛四聯(lián)癥是圓錐動(dòng)脈干畸形最常見的類型,約占所有先心病的12%,基本病理改變?yōu)橛沂伊鞒龅拦Ｗ�、室間隔缺損、主動(dòng)脈騎跨及右心室肥厚。研究其發(fā)病機(jī)制有利于預(yù)防、產(chǎn)前診斷及早期干預(yù),對(duì)于減輕社會(huì)家庭精神、經(jīng)濟(jì)負(fù)擔(dān)有重要意義。目前普遍接受的觀點(diǎn)為先心病是內(nèi)在遺傳易感因素和外界環(huán)境致畸因素共同作用的結(jié)果。先心病候選基因的研究是目前先心病發(fā)病機(jī)制研究的熱點(diǎn)。圓錐動(dòng)脈干畸形是遺傳綜合癥DiGeroge綜合癥(DGS)的主要心臟表現(xiàn),研究發(fā)現(xiàn),80%-100%DGS患者伴染色體22q11.2的單倍體微缺失。目前該區(qū)域已分離出20-30個(gè)候選基因,HIRA基因位于該區(qū)域,作為DGS的候選基因之一,受到廣泛關(guān)注。在動(dòng)物實(shí)驗(yàn)中,下調(diào)雞胚HIRA在神經(jīng)嵴細(xì)胞中表達(dá),90%雞胚發(fā)生永存動(dòng)脈干畸形；下調(diào)小鼠HIRA基因表達(dá),小鼠心臟袢化失敗。法洛氏四聯(lián)癥作為最常見的圓錐動(dòng)脈干畸形,也是22q11.2微缺失的常見表型。據(jù)此推測(cè),HIRA基因可能是TOF的候選基因。但是人類TOF與HIRA的相關(guān)研究非常有限。本課題組前期通過TOF患兒右室流出道心肌HIRA基因表達(dá)研究發(fā)現(xiàn)HIRA基因在TOF患兒表達(dá)較正常降低。其表達(dá)降低的原因有待于進(jìn)一步探討。本課題擬對(duì)法四患兒HIRA基因進(jìn)行編碼區(qū)序列分析,探討HIRA蛋白結(jié)構(gòu)變化與TOF發(fā)生的關(guān)系：同時(shí)分析HIRA基因3’UTR區(qū)序列,探尋可能與3’UTR區(qū)結(jié)合的micro RNAs,找出TOF患兒右室流出道心肌HIRA表達(dá)降低的原因。初步解釋HIRA基因在TOF發(fā)病中的作用機(jī)制。第一部分HIRA基因編碼區(qū)序列分析目的：對(duì)中國(guó)TOF患兒中HIRA基因編碼區(qū)進(jìn)行序列分析,探討HIRA基因編碼區(qū)變異與TOF的關(guān)系。方法：收集TOF患兒外周血278例及年齡和性別與病例組盡量匹配的健康兒童外周血500例,提取基因組DNA,對(duì)HIRA基因全部編碼區(qū)進(jìn)行測(cè)序,突變位點(diǎn)病例組擴(kuò)大到500例,對(duì)氨基酸的改變進(jìn)行生物信息學(xué)預(yù)測(cè),將野生型HIRA基因mRNA與突變后mRNA注入斑馬魚單細(xì)胞時(shí)期后觀察斑馬魚表型進(jìn)行突變功能驗(yàn)證。結(jié)果：1.在12外顯子發(fā)現(xiàn)1個(gè)已報(bào)導(dǎo)的SNP c.G1339GA (rs150603624),其發(fā)生頻率與NCBI dbSNP數(shù)據(jù)庫(kù)報(bào)導(dǎo)的頻率無統(tǒng)計(jì)學(xué)差異。2.第17外顯子發(fā)現(xiàn)1個(gè)突變c.C2301CA,頻率為1/500,用生物信息學(xué)網(wǎng)站預(yù)測(cè)發(fā)現(xiàn)其氨基酸改變對(duì)其蛋白質(zhì)功能影響不大,在物種間保守性不高,推測(cè)該突變可能為良性突變。3.第23外顯子發(fā)現(xiàn)1個(gè)突變c.C3026CT,頻率為2/500,生物信息學(xué)網(wǎng)站預(yù)測(cè)發(fā)現(xiàn)其氨基酸改變對(duì)其蛋白質(zhì)功能影響大,突變位點(diǎn)在不同特種間高度保守,推測(cè)該突變可能為致病性突變,突變mRNA注射后斑馬魚的心臟畸形發(fā)生率明顯高于野生型。小結(jié)：HIRA基因23外顯子突變c.C3026CT可能為致病性突變,可能導(dǎo)致先心病的發(fā)生。第二部分HIRA基因3'UTR序列分析及相關(guān)micro RNAs研究目的：對(duì)中國(guó)TOF患兒HIRA基因3'UTR進(jìn)行序列分析,探討HIRA基因3'UTR序列變異對(duì)調(diào)控其micro RNAs的影響。方法：收集TOF患兒外周血278例及年齡和性別與病例組盡量匹配的健康兒童外周血500例,提取基因組DNA,對(duì)HIRA3'UTR進(jìn)行測(cè)序,用生物信息學(xué)網(wǎng)站預(yù)測(cè)SNP位點(diǎn)周圍的micro RNAs,用雙熒光素酶報(bào)告基因檢測(cè)系統(tǒng)及real-timePCR檢測(cè)micro RNA與HIRA 3'UTR的相互作用。結(jié)果：1.HIRA基因3'UTR SNP(rs:117447448)位點(diǎn)的分布在病例組和對(duì)照組中存在統(tǒng)計(jì)學(xué)差異(P=0.001)。2.在線預(yù)測(cè)SNP(rs:117447448)位點(diǎn)上游8 bp存在microRNA 328的結(jié)合位點(diǎn)。3.共同轉(zhuǎn)染p-silencer+miR328質(zhì)粒和pgl3-promoter+HIRA3'UTR質(zhì)粒293細(xì)胞熒光素酶的活性(0.0125±0.0006)低于單獨(dú)轉(zhuǎn)染pgl3-promoter+HIRA3'UTR質(zhì)粒的活性(0.01963±0.0038),其差異有統(tǒng)計(jì)學(xué)意義(P=0.0327)。轉(zhuǎn)染p-silencer+miR328質(zhì)粒的293T細(xì)胞HIRA基因的表達(dá)(1.03961±0.0772)較轉(zhuǎn)染p-silencer空載質(zhì)粒的表達(dá)(1.60865±0.2749)降低,差異有統(tǒng)計(jì)學(xué)意義(P=-0.037)。4.用SNP(rs:117447448)位點(diǎn)不同基因型的質(zhì)粒與miR328共同轉(zhuǎn)染293細(xì)胞,不同基因型的熒光素酶活性無統(tǒng)計(jì)學(xué)差異(P=0.38)小結(jié)HIRA基因3'UTR區(qū)rs:117447448SNP位點(diǎn)可能與TOF的發(fā)生相關(guān)：HIRA基因是micro RNA328的靶基因。rs:117447448SNP位點(diǎn)不是micro RNA328作用于HIRA基因的主要靶位。
[Abstract]:Congenital heart disease (congenital heart disease) is a congenital malformation caused by abnormal development of the heart and large blood vessels in the fetal stage. The incidence of congenital heart disease is 6%0-10%0. in live born infants, which is about 150 thousand children born with congenital heart disease every year in China, causing serious mental and economic burdens in social families. Intractable hypoxemia, untreated, the early death rate up to 80%. conical artery stem deformities 60%-70%. Fallot is the most common type of conical artery stem deformity, accounting for about 12% of all congenital heart disease. The basic pathological changes are right ventricular outflow tract obstruction, ventricular septal defect, aortic riding span and right ventricular hypertrophy. Its pathogenesis is beneficial to prevention, prenatal diagnosis and early intervention are of great significance for reducing social family spirit and economic burden. At present, it is widely accepted that congenital heart disease is the result of inherent genetic susceptibility factors and external environment teratogenic factors. The study of candidate genes of congenital heart disease is the study of the pathogenesis of congenital heart disease. Conical artery stem deformity is the main cardiac manifestation of genetic syndrome DiGeroge syndrome (DGS). The study found that 80%-100%DGS patients with chromosome 22q11.2 haploid microdeletion. 20-30 candidate genes have been isolated in this region. The HIRA gene is located in the region and is one of the candidate genes for DGS. In the experiment, the down regulated chicken embryo HIRA was expressed in the neural crest cells, and the 90% chicken embryo had persistent arteriosk deformity; the HIRA gene was downregulated and the heart loop of the mice failed. The Fallot tetralogy was the most common conical artery stem deformity and the common phenotype of 22q11.2 microdeletion. According to this, the HIRA gene may be a candidate gene of TOF. The related research of human TOF and HIRA is very limited. In the earlier period, we found that the expression of HIRA gene in the right ventricular outflow tract of children with TOF was lower than normal in the right ventricular outflow tract of children with the HIRA gene expression. The reason for the decrease of the expression of the HIRA gene is to be further discussed. This subject is intended to analyze the sequence of the coding region of the HIRA gene in four children of France and explore HIRA. The relationship between the changes of protein structure and the occurrence of TOF: simultaneous analysis of the 3 'UTR region of the HIRA gene and the micro RNAs that may be combined with the 3' UTR region to find out the decrease of HIRA expression in the right ventricular outflow tract in children with TOF. The preliminary interpretation of the mechanism of the HIRA gene in the pathogenesis of TOF. The sequence analysis of the coding region of HIRA gene in children with TOF was carried out to investigate the relationship between the mutation of the HIRA gene coding region and the TOF. Methods: 278 cases of peripheral blood of children with TOF and 500 cases of healthy children's peripheral blood which were matched with age and sex and case group were collected, the genomic DNA was extracted, the whole coding region of the HIRA gene was sequenced, and the case group of the mutation site was enlarged. In 500 cases, the changes of amino acids were predicted by bioinformatics. The mutant function of the wild type HIRA gene mRNA and the mutant mRNA was observed after the single cell period of zebrafish. Results: 1. the 1 reported SNP c.G1339GA (rs150603624) was found in exon 12, and the frequency of its occurrence and the NCBI dbSNP Database Report The frequency of.2. seventeenth exon found 1 mutations c.C2301CA, the frequency was 1/500, and the bioinformatics website predicted that the amino acid changes had little influence on its protein function, and the conservatism between species was not high. It was presumed that the mutation may be a benign mutation of.3. twenty-third exon 1 mutation c.C3026CT, the frequency of 2/500, birth rate. The prediction of the information science website found that the amino acid changes have great influence on its protein function, and the mutation site is highly conserved in different special rooms. It is suggested that the mutation may be a pathogenic mutation. The incidence of heart malformation of zebrafish after mRNA injection is obviously higher than that of the wild type. The mutation of HIRA gene 23 exon mutation c.C3026CT may be pathogenicity. Mutation, may lead to the occurrence of congenital heart disease. Second part HIRA gene 3'UTR sequence analysis and related micro RNAs research purposes: sequence analysis of HIRA gene 3'UTR in children with TOF in Chinese children, to explore the effect of the 3'UTR sequence variation of HIRA gene on the regulation of micro RNAs. Methods: 278 cases of peripheral blood and age, sex and case group were collected. 500 cases of peripheral blood of healthy children were matched, genomic DNA was extracted, HIRA3'UTR was sequenced, micro RNAs around SNP site was predicted by bioinformatics website, and the interaction between micro RNA and HIRA 3'UTR was detected by double luciferase reporter gene detection system and real-timePCR. The distribution in the case group and the control group was statistically different (P=0.001).2. on-line prediction of the SNP (rs:117447448) site upstream 8 bp existence microRNA 328 binding site.3. co transfection p-silencer+miR328 plasmids and pgl3-promoter+HIRA3'UTR plasmid 293 cell luciferase activity (0.0125 + 0.0006) lower than the single transfection of pgl3-promoter+HIRA. The activity of 3'UTR plasmid was (0.01963 + 0.0038), and the difference was statistically significant (P=0.0327). The expression of HIRA gene in 293T cells transfected with p-silencer+miR328 plasmid (1.03961 + 0.0772) was lower than that of transfected p-silencer empty plasmid (1.60865 + 0.2749), and the difference was statistically significant (P=-0.037).4. with the different genotype of SNP (rs:117447448) loci. The plasmids and miR328 co transfected 293 cells, and there was no significant difference in the luciferase activity of different genotypes (P=0.38). The rs:117447448SNP locus in 3'UTR region of HIRA gene may be related to the occurrence of TOF. The HIRA gene is the target gene of micro RNA328, which is not the main target of micro RNA328.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R725.4

【共引文獻(xiàn)】

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本文編號(hào)：2103320