MUTYH基因AluYb8MUTYH多态性对人线粒体DNA（mtDNA）影响的研究

发布时间：2018-05-07 17:22

本文选题：MUTYH基因 + 线粒体DNA　；参考：《南京大学》2012年硕士论文

【摘要】：实验一AluYb8MUTYH多态性与线粒体D-LOOP区突变谱带关系的分析背景与目的：在多种mtDNA修复机制中,碱基切除修复(BER)是最为普遍的修复方式。MUTYH基因在BER系统中占有主要的作用,与APE1协同作用切除与8-OHdG错配的A,形成AP位点,进行基因的长片段修复(long patch repair),阻止基因组中G:C→T:A的突变。MUTYH基因编码的蛋白存在不同的亚细胞定位,Ⅰ型蛋白产物定位于线粒体内,维持线粒体基因组的稳定；Ⅱ型蛋白产物定位于细胞核内,维持核内基因组的稳定。本研究着重分析导致MUTYH基因Ⅰ型蛋白产物表达受抑的AluYb8MUTYH与mtDNA LOOP区突变谱型的关系。方法：以60例正常人群为研究对象,从外周血中提取DNA,采用PCR-琼脂糖凝胶电泳方法进行AluYb8MUTYH多态性基因分型,电泳分析显示,MUTYH基因intron15呈现出二种不同的片段,长片段为800多bp,含AIuYb8插入序列,短片段为500bp；人群中存在三种基因型,分别为纯合插入型(AluYb8presence/presence, P/P)、纯合野生型(AluYb8absence/absence, A/A)以及杂合型(AluYb8presence/absence, A/P)。同时采用PCR技术扩增线粒体D-LOOP区DNA, T载体克隆PCR产物,并从含目的片段的菌液提取质粒,每例样本选取10个克隆质粒进行测序,比对不同克隆质粒测序结果,分析AluYb8MUTYH不同基因型个体D-LOOP区突变谱带。结果：共发现350个变异位点,大部分变异类型为碱基置换(67%),同时也有短串联重复序列的变异(short tandem repeats,STRs)和碱基缺失／插入,占总变异的29%和4%。P/P基因型平均碱基变异率低于A/A基因型和A/P基因型,但各基因型间比较无统计学差异。结论：MUTYH基因AluYb8插入变异与人mtDNA突变率无显著关系。实验二2型糖尿病患者外周血细胞线粒体拷贝数与AluYb8MUTYH多态性变异的相关性研究背景与目的：2型糖尿病是以高血糖为特征的一种胰岛素抵抗状态,是威胁人类健康的主要疾病之一。糖尿病的发生发展与氧化应激密切相关,高血糖可以引起自由基的产生,导致DNA氧化损伤产物的累积。2型糖尿患者中,8-OHdG是最常见的DNA氧化损伤产物。这种以8-OHdG为标志的DNA氧化损伤主要是通过碱基切除修复机制(base excision repair, BER)来修复。许多研究已确定BER系统基因(hOGG1, MUTYH)变异与2型糖尿病的发病易感性之间的关系。本实验室前期工作发现,人类MUTYH基因第15内含子中存在AluYb8插入变异(AluYb8MUTYH),在中国人群中呈多态性分布。病例-对照研究已显示AluYb8MUTYH P/P基因型是2型糖尿病发病的一个危险因素,本研究进一步探讨AluYb8hMUTYH P/P基因型增加2型糖尿病发病风险可能存在的线粒体机制。研究方法：采集125例2型糖尿病患者和146例健康对照的外周血,提取外周血白细胞DNA,鉴定各样本AluYb8MUTYH基因型。应用荧光实时定量PCR测定入选病例组及对照组个体外周血中mtDNA含量。运用SPSS16.0软件分析糖尿病患者中AluYb8MUTYH不同基因型个体mtDNA含量的差异,同时分析比较携带(AluYb8MUTYH)相同基因型的2型糖尿病患者与健康对照mtDNA含量的变化。结果：携带P/P基因型健康人群外周血mtDNA含量低于携带A/A基因型人群。小于60岁的2型糖尿病组,P/P基因型mtDNA含量显著高于A/P基因型和A/A基因型,这一变化在大于60岁年龄组未发现。与正常对照相比,携带P/P基因型2型糖尿病组外周血mtDNA含量显著高于正常对照组,携带A/P和A/A基因型2型糖尿病患者与正常对照相比,外周血mtDNA含量均无统计学差异。结论：AluYb8MUTYH P/P基因型作为2型糖尿病发病的一个危险因素,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.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R394

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