线粒体DNA常见缺失在大片段缺失突变中的作用及其发生机制

发布时间：2018-04-11 07:28

本文选题：D-半乳糖 + 线粒体DNA　；参考：《华中科技大学》2011年博士论文

【摘要】：第一部分D-半乳糖诱导大鼠内耳线粒体DNA大片段缺失突变的研究目的：研究D-半乳糖模型大鼠内耳组织中可能存在的线粒体DNA大片段缺失突变。方法：1月龄雌性Wistar大鼠48只随机分为4组：低、中、高剂量D-半乳糖组和生理盐水对照组各12只,分别于每日皮下注射5%D-半乳糖150 mg/kg、300 mg/kg、500 mg/kg和等体积生理盐水,共8周。听性脑干反应(auditory brainstem response,ABR)检测大鼠听阈。联合应用普通PCR、巢式PCR和长片段PCR技术筛查大鼠内耳组织中的线粒体DNA大片段缺失突变。应用DNA测序技术证实线粒体DNA缺失突变的存在。结果：D-半乳糖组大鼠与对照组之间ABR阈移差异无统计学意义(p0.05)。不同剂量D-半乳糖组大鼠内耳组织中均可检测出线粒体DNA 4834 bp缺失突变,并且首次发现3种新的线粒体DNA缺失突变。线粒体DNA 4834 bp缺失突变和3种新发现的缺失突变的断裂融合位点两端均存在核苷酸重复序列,并且均涉及线粒体编码的细胞色素C氧化酶亚基Ⅲ(COX3)基因。结论：D-半乳糖模型大鼠内耳组织中存在多种线粒体DNA大片段缺失突变,这些缺失突变存在相似的特征。第二部分D-半乳糖大鼠内耳线粒体DNA 4834 bp缺失在大片段缺失突变中作用的研究目的：探讨线粒体DNA 4834 bp缺失突变在大片段缺失突变中的贡献率,以及大片段缺失突变对缺失突变热点区域的细胞色素C氧化酶(COX)活性的影响。方法：应用Taqman探针法实时定量PCR技术检测不同剂量D-半乳糖组大鼠内耳组织的线粒体DNA 4834 bp缺失突变率、线粒体DNA大片段缺失突变率及线粒体DNA拷贝数目。比色法定量检测细胞色素C氧化酶(COX)活性变化。结果：与对照组相比,三个不同剂量D-半乳糖组大鼠内耳组织中线粒体DNA4834 bp缺失突变率和线粒体DNA大片段缺失突变率明显增高(p0.05),其中高剂量D-半乳糖组升高最明显。低、中、高剂量D-半乳糖组大鼠内耳组织中线粒体DNA4834 bp缺失在线粒体DNA大片段缺失突变中所占比例逐渐减少,分别为76.71±1.96%,74.94±2.04%,67.86±2.38%,而对照组为87.74土3.52%(p0.05)；D-半乳糖各剂量组内耳组织的线粒体拷贝数目与对照组相比均明显增高(p0.05),并随D-半乳糖剂量增大而显著增高；COX活性与对照组相比均明显降低,并随D-半乳糖剂量增大而显著降低。结论：线粒体DNA 4834 bp缺失突变是D-半乳糖大鼠模型内耳组织中的一种最主要的线粒体DNA大片段缺失。此缺失突变可考虑作为评估内耳组织线粒体DNA损伤的理想的分子标志物。第三部分D-半乳糖大鼠内耳线粒体DNA缺失突变的发生机制研究目的：探讨大鼠内耳组织线粒体DNA缺失突变可能的发生机制。方法：88只雄性Wistar大鼠随机分为4组：低、中、高剂量D-半乳糖组和生理盐水对照组各12只,分别于每日皮下注射5%D-半乳糖150 mg/kg、300 mg/kg、500 mg/kg和等体积生理盐水,共8周。应用实时定量PCR技术检测各组大鼠内耳组织中线粒体DNA 4834 bp缺失突变率和线粒体DNA相对拷贝数目。应用实时定量逆转录PCR技术和Western blotting方法检测各组大鼠内耳组织中线粒体转录因子A(mitochondrial transcription factor A, TFAM)、DNA聚合酶y (DNA polymerase y, DNA pol y)和DNA修复酶8-氧鸟嘌呤糖苷酶(8-oxoguanine DNA glycosylase, OGG1)在基因和蛋白水平的变化。结果：与对照组相比,D-半乳糖组大鼠内耳组织中线粒体DNA 4834 bp缺失率和线粒体DNA相对拷贝数目明显增高(p0.05),高剂量组升高最明显。D-半乳糖各剂量组内耳组织中DNA pol y和OGG1的基因和蛋白表达水平均明显低于对照组(p0.05),并随D-半乳糖剂量增大而明显降低。各剂量组内耳组织中TFAM的基因和蛋白表达水平与对照组相比均明显增高(p0.05),并随D-半乳糖剂量增大而明显增高。结论：在内耳老化过程中线粒体DNA缺失突变的积累可能是由于线粒体DNA修复功能减退和TFAM过表达所致的线粒体DNA复制增加造成的。
[Abstract]:The first part of D- galactose induced deletion mutation of large fragment of mitochondrial DNA fragment in the inner ear of rats
Objective: To study the deletion mutation of large fragment of mitochondrial DNA fragment that may exist in the inner ear tissue of D- galactose model rats.
Methods: 1 month old female Wistar 48 rats were randomly divided into 4 groups: low, high dose of D- galactose group and saline control group with 12 rats in each group, respectively in the subcutaneous injection of 5%D- galactose 150 mg/kg, 300 mg/kg, 500 mg/kg and normal saline for 8 weeks. The auditory brainstem response (auditory brainstem response, ABR) were detected. The combined application of PCR threshold, deletion of mitochondrial DNA fragment of inner ear tissue nested PCR and PCR long fragment screening technology in rats. Mutations by DNA sequencing confirmed that mitochondrial DNA deletion mutations.
Results: D- galactose group rats and control group ABR threshold shift was no statistically significant difference (P0.05). All different doses of D- in D-galactose rats inner ear tissue detected in the mitochondrial DNA 4834 bp deletion mutation, and discovered that 3 mutations of mitochondrial DNA deletion. The new mitochondrial DNA 4834 bp deletion mutation and 3 a newly discovered mutation deletion breakpoints are two nucleotide repeat sequences, cytochrome C oxidase subunit III and are involved in mitochondrial encoding gene (COX3).
Conclusion: there are many large deletion mutations of mitochondrial DNA fragments in the inner ear tissue of D- galactose model rats. These missing mutations have similar characteristics.
Study on the role of mitochondrial DNA 4834 bp deletion in the inner ear of D- galactose rats in the deletion mutation of large fragment
Objective: To investigate the contribution rate of mitochondrial DNA 4834 bp deletion mutation in large fragment deletion mutation, and the effect of large deletion mutation on the activity of cytochrome C oxidase (COX) in the hotspot of deletion mutation.
Methods: the application of Taqman method to detect mitochondrial DNA probe real-time quantitative PCR technique with different doses of D- galactose group in the rat inner ear tissue 4834 bp deletion mutation rate, mitochondrial DNA large deletion mutation rate and mitochondrial DNA copy number. Colorimetric method for quantitative detection of cytochrome C oxidase (COX) activity.
Results: compared with control group, large deletion mutation rate significantly increased mitochondrial DNA4834 and mitochondrial DNA bp deletion mutation rate of three different doses of D- galactose group in the rat inner ear tissues (P0.05), the high dose of D- galactose group was increased significantly. The low, inner ear tissue in rats of high dose D- lactose in mitochondrial DNA4834 bp deletion in mitochondrial DNA gradually decreased for large deletion mutations in the proportion were 76.71 + 1.96%, 74.94 + 2.04%, 67.86 + 2.38%, and 87.74 in the control group 3.52% (P0.05); D- galactose in each dose group of inner ear tissue mitochondrial copy number were significantly increased compared with the control group (P0.05), and with the D- galactose dose increases significantly increased; the activity of COX were significantly decreased compared with the control group, and with the D- galactose dose decreased.
Conclusion: mitochondrial DNA 4834 bp deletion mutation is a major mitochondrial DNA deletion in the D- inner ear tissues of rats. This deletion mutation can be considered as an ideal molecular marker to evaluate mitochondrial DNA damage in inner ear tissues.
Study on the mechanism of mitochondrial DNA deletion mutation in the inner ear of D- galactose rats
Objective: To investigate the possible mechanism of mitochondrial DNA deletion mutation in the inner ear tissue of rats.
Methods: 88 male Wistar rats were randomly divided into 4 groups: low, high dose of D- galactose group and saline control group with 12 rats in each group, respectively in the subcutaneous injection of 5%D- galactose 150 mg/kg, 300 mg/kg, 500 mg/kg and normal saline for 8 weeks. The mitochondrial DNA detected the rat inner ear tissue using real-time quantitative PCR technique in 4834 bp deletion mutation rate and mitochondrial DNA relative copy number. Mitochondrial transcription factor A were detected in rat inner ear tissue using real-time quantitative reverse transcription PCR and Western blotting method (mitochondrial transcription factor A, TFAM), y (DNA polymerase y DNA polymerase, DNA pol y) DNA and 8- repair enzyme yguanine glycosidase (8-oxoguanine DNA glycosylase, OGG1) changes in gene and protein level.
Results: compared with control group, the mitochondrial DNA inner ear tissue in rats of D- galactose 4834 bp deletion rate and relative copy number of mitochondrial DNA increased significantly (P0.05), high dose group increased DNA gene and protein of Pol y and OGG1.D- as the most significant galactose groups in the inner ear tissues expression levels were significantly lower than the control group (P0.05), and with the D- galactose dose decreases. TFAM gene and protein in each dose group of inner ear tissue the expression level were significantly increased compared with the control group (P0.05), and with the increasing doses of D- galactose increased significantly.
Conclusion: the accumulation of mitochondrial DNA deletion may be caused by mitochondrial DNA repair dysfunction and mitochondrial DNA replication induced by over expression of TFAM in the aging process of inner ear.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346

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