本文選題：D-半乳糖 + 線粒體DNA��； 參考：《華中科技大學(xué)》2011年博士論文

【摘要】：第一部分D-半乳糖誘導(dǎo)大鼠內(nèi)耳線粒體DNA大片段缺失突變的研究 目的：研究D-半乳糖模型大鼠內(nèi)耳組織中可能存在的線粒體DNA大片段缺失突變。 方法：1月齡雌性Wistar大鼠48只隨機(jī)分為4組：低、中、高劑量D-半乳糖組和生理鹽水對照組各12只,分別于每日皮下注射5%D-半乳糖150 mg/kg、300 mg/kg、500 mg/kg和等體積生理鹽水,共8周。聽性腦干反應(yīng)(auditory brainstem response,ABR)檢測大鼠聽閾。聯(lián)合應(yīng)用普通PCR、巢式PCR和長片段PCR技術(shù)篩查大鼠內(nèi)耳組織中的線粒體DNA大片段缺失突變。應(yīng)用DNA測序技術(shù)證實線粒體DNA缺失突變的存在。 結(jié)果：D-半乳糖組大鼠與對照組之間ABR閾移差異無統(tǒng)計學(xué)意義(p0.05)。不同劑量D-半乳糖組大鼠內(nèi)耳組織中均可檢測出線粒體DNA 4834 bp缺失突變,并且首次發(fā)現(xiàn)3種新的線粒體DNA缺失突變。線粒體DNA 4834 bp缺失突變和3種新發(fā)現(xiàn)的缺失突變的斷裂融合位點兩端均存在核苷酸重復(fù)序列,并且均涉及線粒體編碼的細(xì)胞色素C氧化酶亞基Ⅲ(COX3)基因。 結(jié)論：D-半乳糖模型大鼠內(nèi)耳組織中存在多種線粒體DNA大片段缺失突變,這些缺失突變存在相似的特征。 第二部分D-半乳糖大鼠內(nèi)耳線粒體DNA 4834 bp缺失在大片段缺失突變中作用的研究 目的：探討線粒體DNA 4834 bp缺失突變在大片段缺失突變中的貢獻(xiàn)率,以及大片段缺失突變對缺失突變熱點區(qū)域的細(xì)胞色素C氧化酶(COX)活性的影響。 方法：應(yīng)用Taqman探針法實時定量PCR技術(shù)檢測不同劑量D-半乳糖組大鼠內(nèi)耳組織的線粒體DNA 4834 bp缺失突變率、線粒體DNA大片段缺失突變率及線粒體DNA拷貝數(shù)目。比色法定量檢測細(xì)胞色素C氧化酶(COX)活性變化。 結(jié)果：與對照組相比,三個不同劑量D-半乳糖組大鼠內(nèi)耳組織中線粒體DNA4834 bp缺失突變率和線粒體DNA大片段缺失突變率明顯增高(p0.05),其中高劑量D-半乳糖組升高最明顯。低、中、高劑量D-半乳糖組大鼠內(nèi)耳組織中線粒體DNA4834 bp缺失在線粒體DNA大片段缺失突變中所占比例逐漸減少,分別為76.71±1.96%,74.94±2.04%,67.86±2.38%,而對照組為87.74土3.52%(p0.05)；D-半乳糖各劑量組內(nèi)耳組織的線粒體拷貝數(shù)目與對照組相比均明顯增高(p0.05),并隨D-半乳糖劑量增大而顯著增高；COX活性與對照組相比均明顯降低,并隨D-半乳糖劑量增大而顯著降低。 結(jié)論：線粒體DNA 4834 bp缺失突變是D-半乳糖大鼠模型內(nèi)耳組織中的一種最主要的線粒體DNA大片段缺失。此缺失突變可考慮作為評估內(nèi)耳組織線粒體DNA損傷的理想的分子標(biāo)志物。 第三部分D-半乳糖大鼠內(nèi)耳線粒體DNA缺失突變的發(fā)生機(jī)制研究 目的：探討大鼠內(nèi)耳組織線粒體DNA缺失突變可能的發(fā)生機(jī)制。 方法：88只雄性Wistar大鼠隨機(jī)分為4組：低、中、高劑量D-半乳糖組和生理鹽水對照組各12只,分別于每日皮下注射5%D-半乳糖150 mg/kg、300 mg/kg、500 mg/kg和等體積生理鹽水,共8周。應(yīng)用實時定量PCR技術(shù)檢測各組大鼠內(nèi)耳組織中線粒體DNA 4834 bp缺失突變率和線粒體DNA相對拷貝數(shù)目。應(yīng)用實時定量逆轉(zhuǎn)錄PCR技術(shù)和Western blotting方法檢測各組大鼠內(nèi)耳組織中線粒體轉(zhuǎn)錄因子A(mitochondrial transcription factor A, TFAM)、DNA聚合酶y (DNA polymerase y, DNA pol y)和DNA修復(fù)酶8-氧鳥嘌呤糖苷酶(8-oxoguanine DNA glycosylase, OGG1)在基因和蛋白水平的變化。 結(jié)果：與對照組相比,D-半乳糖組大鼠內(nèi)耳組織中線粒體DNA 4834 bp缺失率和線粒體DNA相對拷貝數(shù)目明顯增高(p0.05),高劑量組升高最明顯。D-半乳糖各劑量組內(nèi)耳組織中DNA pol y和OGG1的基因和蛋白表達(dá)水平均明顯低于對照組(p0.05),并隨D-半乳糖劑量增大而明顯降低。各劑量組內(nèi)耳組織中TFAM的基因和蛋白表達(dá)水平與對照組相比均明顯增高(p0.05),并隨D-半乳糖劑量增大而明顯增高。 結(jié)論：在內(nèi)耳老化過程中線粒體DNA缺失突變的積累可能是由于線粒體DNA修復(fù)功能減退和TFAM過表達(dá)所致的線粒體DNA復(fù)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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R346

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