本文選題：MUTYH基因 + 線粒體DNA��； 參考：《南京大學(xué)》2012年碩士論文

【摘要】：實(shí)驗(yàn)一AluYb8MUTYH多態(tài)性與線粒體D-LOOP區(qū)突變譜帶關(guān)系的分析 背景與目的：在多種mtDNA修復(fù)機(jī)制中,堿基切除修復(fù)(BER)是最為普遍的修復(fù)方式。MUTYH基因在BER系統(tǒng)中占有主要的作用,與APE1協(xié)同作用切除與8-OHdG錯(cuò)配的A,形成AP位點(diǎn),進(jìn)行基因的長(zhǎng)片段修復(fù)(long patch repair),阻止基因組中G:C→T:A的突變。MUTYH基因編碼的蛋白存在不同的亞細(xì)胞定位,Ⅰ型蛋白產(chǎn)物定位于線粒體內(nèi),維持線粒體基因組的穩(wěn)定；Ⅱ型蛋白產(chǎn)物定位于細(xì)胞核內(nèi),維持核內(nèi)基因組的穩(wěn)定。本研究著重分析導(dǎo)致MUTYH基因Ⅰ型蛋白產(chǎn)物表達(dá)受抑的AluYb8MUTYH與mtDNA LOOP區(qū)突變譜型的關(guān)系。 方法：以60例正常人群為研究對(duì)象,從外周血中提取DNA,采用PCR-瓊脂糖凝膠電泳方法進(jìn)行AluYb8MUTYH多態(tài)性基因分型,電泳分析顯示,MUTYH基因intron15呈現(xiàn)出二種不同的片段,長(zhǎng)片段為800多bp,含AIuYb8插入序列,短片段為500bp；人群中存在三種基因型,分別為純合插入型(AluYb8presence/presence, P/P)、純合野生型(AluYb8absence/absence, A/A)以及雜合型(AluYb8presence/absence, A/P)。同時(shí)采用PCR技術(shù)擴(kuò)增線粒體D-LOOP區(qū)DNA, T載體克隆PCR產(chǎn)物,并從含目的片段的菌液提取質(zhì)粒,每例樣本選取10個(gè)克隆質(zhì)粒進(jìn)行測(cè)序,比對(duì)不同克隆質(zhì)粒測(cè)序結(jié)果,分析AluYb8MUTYH不同基因型個(gè)體D-LOOP區(qū)突變譜帶。 結(jié)果：共發(fā)現(xiàn)350個(gè)變異位點(diǎn),大部分變異類(lèi)型為堿基置換(67%),同時(shí)也有短串聯(lián)重復(fù)序列的變異(short tandem repeats,STRs)和堿基缺失／插入,占總變異的29%和4%。P/P基因型平均堿基變異率低于A/A基因型和A/P基因型,但各基因型間比較無(wú)統(tǒng)計(jì)學(xué)差異。 結(jié)論：MUTYH基因AluYb8插入變異與人mtDNA突變率無(wú)顯著關(guān)系。 實(shí)驗(yàn)二2型糖尿病患者外周血細(xì)胞線粒體拷貝數(shù)與AluYb8MUTYH多態(tài)性變異的相關(guān)性研究 背景與目的：2型糖尿病是以高血糖為特征的一種胰島素抵抗?fàn)顟B(tài),是威脅人類(lèi)健康的主要疾病之一。糖尿病的發(fā)生發(fā)展與氧化應(yīng)激密切相關(guān),高血糖可以引起自由基的產(chǎn)生,導(dǎo)致DNA氧化損傷產(chǎn)物的累積。2型糖尿患者中,8-OHdG是最常見(jiàn)的DNA氧化損傷產(chǎn)物。這種以8-OHdG為標(biāo)志的DNA氧化損傷主要是通過(guò)堿基切除修復(fù)機(jī)制(base excision repair, BER)來(lái)修復(fù)。許多研究已確定BER系統(tǒng)基因(hOGG1, MUTYH)變異與2型糖尿病的發(fā)病易感性之間的關(guān)系。本實(shí)驗(yàn)室前期工作發(fā)現(xiàn),人類(lèi)MUTYH基因第15內(nèi)含子中存在AluYb8插入變異(AluYb8MUTYH),在中國(guó)人群中呈多態(tài)性分布。病例-對(duì)照研究已顯示AluYb8MUTYH P/P基因型是2型糖尿病發(fā)病的一個(gè)危險(xiǎn)因素,本研究進(jìn)一步探討AluYb8hMUTYH P/P基因型增加2型糖尿病發(fā)病風(fēng)險(xiǎn)可能存在的線粒體機(jī)制。 研究方法：采集125例2型糖尿病患者和146例健康對(duì)照的外周血,提取外周血白細(xì)胞DNA,鑒定各樣本AluYb8MUTYH基因型。應(yīng)用熒光實(shí)時(shí)定量PCR測(cè)定入選病例組及對(duì)照組個(gè)體外周血中mtDNA含量。運(yùn)用SPSS16.0軟件分析糖尿病患者中AluYb8MUTYH不同基因型個(gè)體mtDNA含量的差異,同時(shí)分析比較攜帶(AluYb8MUTYH)相同基因型的2型糖尿病患者與健康對(duì)照mtDNA含量的變化。 結(jié)果：攜帶P/P基因型健康人群外周血mtDNA含量低于攜帶A/A基因型人群。小于60歲的2型糖尿病組,P/P基因型mtDNA含量顯著高于A/P基因型和A/A基因型,這一變化在大于60歲年齡組未發(fā)現(xiàn)。與正常對(duì)照相比,攜帶P/P基因型2型糖尿病組外周血mtDNA含量顯著高于正常對(duì)照組,攜帶A/P和A/A基因型2型糖尿病患者與正常對(duì)照相比,外周血mtDNA含量均無(wú)統(tǒng)計(jì)學(xué)差異。 結(jié)論：AluYb8MUTYH P/P基因型作為2型糖尿病發(fā)病的一個(gè)危險(xiǎn)因素,可增加2型糖尿病患者外周血mtDNA含量。
[Abstract]:Analysis of the relationship between polymorphism of AluYb8MUTYH and mutation band of mitochondrial D-LOOP region in Experiment 1
Background and purpose: in a variety of mtDNA repair mechanisms, base excision repair (BER) is the most common repair method,.MUTYH gene plays a major role in the BER system. In collaboration with APE1, the removal of A, 8-OHdG mismatched A, a AP site, and a long fragment repair of the gene (long patch repair), to prevent the mutation of the genome in the genome. The protein encoded by the TYH gene has different subcellular localization. The type I protein product is located in the mitochondria to maintain the stability of the mitochondrial genome. The product of type II protein is located in the nucleus and maintains the stability of the nuclear genome. This study focuses on the analysis of the AluYb8MUTYH and mtDNA LOOP that lead to the inhibition of the expression of MUTYH gene type I egg white products. The relationship between the mutation spectrum type of the region.
Methods: the DNA was extracted from the peripheral blood of 60 normal people, and the PCR- agarose gel electrophoresis was used to classify the AluYb8MUTYH polymorphism. The electrophoresis analysis showed that the MUTYH gene intron15 showed two different fragments, the long fragment was more than 800 BP, the AIuYb8 insertion sequence and the short fragment were 500bp; there were three species in the population. Genotypes were AluYb8presence/presence (P/P), homozygous wild type (AluYb8absence/absence, A/A) and heterozygous type (AluYb8presence/absence, A/P). PCR technology was used to amplify DNA in mitochondrial D-LOOP region, PCR products were cloned by T vector, and plasmid was extracted from the liquid containing the target fragment, and 10 clones were selected for each sample. The plasmid was sequenced and compared with the sequencing results of different clone plasmids. The mutation bands of D-LOOP in different genotypes of AluYb8MUTYH were analyzed.
Results: a total of 350 variation sites were found, most of which were base replacement (67%), and there were also short tandem repeats (short tandem repeats, STRs) and base deletion / insertion, accounting for 29% of the total variation, and the average base variation of 4%.P/P genotypes was lower than that of A/A genotypes and A/P genotypes, but there was no statistical difference among the genotypes. Learning differences.
Conclusion: the AluYb8 insertion mutation of MUTYH gene has no significant correlation with the mutation rate of human mtDNA.
Experimental two. Correlation between mitochondrial copy number of peripheral blood cells and AluYb8MUTYH polymorphism in type 2 diabetic patients
Background and purpose: type 2 diabetes is a state of insulin resistance characterized by hyperglycemia. It is one of the major diseases that threaten human health. The development of diabetes is closely related to oxidative stress. Hyperglycemia can cause the production of free radicals, and 8-OHdG is the most common type of.2 diabetes in patients with the product of DNA oxidative damage. DNA oxidative damage products. The DNA oxidative damage marked by 8-OHdG is mainly repaired by the base excision repair (BER) mechanism. Many studies have identified the relationship between the BER system gene (hOGG1, MUTYH) variation and the susceptibility to type 2 diabetes. Earlier work in the laboratory found that the human MUTYH gene was fifteenth. The AluYb8 insertion mutation (AluYb8MUTYH) exists in the intron and is polymorphic in the Chinese population. Case control study has shown that the AluYb8MUTYH P/P genotype is a risk factor for the onset of type 2 diabetes. This study further explores the possible mitochondrial mechanism of the risk of increasing the risk of type 2 glycan disease by the AluYb8hMUTYH P/P genotype.
Methods: the peripheral blood of 125 patients with type 2 diabetes and 146 healthy controls was collected, the peripheral blood leukocyte DNA was extracted and the AluYb8MUTYH genotypes were identified. The mtDNA content in the selected case group and the control group was measured by real time fluorescence quantitative PCR. The SPSS16.0 soft parts were used to analyze the different AluYb8MUTYH groups in the diabetic patients. Based on the difference of mtDNA content among individuals, the changes of mtDNA content in type 2 diabetes patients with normal genotype (AluYb8MUTYH) and healthy controls were analyzed and compared.
Results: the content of mtDNA in peripheral blood of the healthy people with P/P genotype was lower than that in the group with A/A genotype. The P/P genotype mtDNA content was significantly higher than that of the A/P genotype and A/A genotype in the group of type 2 diabetes less than 60 years old. This change was not found in the age group over 60 years old. Compared with the normal control, the peripheral blood mtDNA was carried in the type 2 diabetes group with P/P genotype. The content of mtDNA in type 2 diabetic patients with A/P and A/A genotype was not significantly different from that in normal controls.
Conclusion: AluYb8MUTYH P/P genotype as a risk factor for type 2 diabetes mellitus can increase mtDNA content in peripheral blood of patients with type 2 diabetes.

【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】：2012
【分類(lèi)號(hào)】：R394

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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本文編號(hào)：1857795