APE1在线粒体DNA损伤修复中的作用及其线粒体定位机制的研究

发布时间：2018-01-07 01:01

本文关键词：APE1在线粒体DNA损伤修复中的作用及其线粒体定位机制的研究　出处：《第三军医大学》2008年硕士论文　论文类型：学位论文

【摘要】： 氧化应激与多种病理状态密切相关,包括癌症,衰老和心血管疾病等。DNA是氧化应激损伤的重要靶分子之一,形成的DNA氧化性损伤主要经碱基切除修复(BER)进行修复。APE1为BER途径的限速酶,其表达水平高低对于BER的活性至关重要。线粒体是除细胞核外唯一具有基因组DNA的细胞器,由于缺乏组蛋白,蛋白编码区密集无内含子,加之与电子转运链相邻,mtDNA极易受到氧化应激的损伤,有研究表明正常细胞mtDNA氧化损伤稳态的水平是nDNA的3-15倍。然而,正常细胞mtDNA修复活性较弱,通过基因治疗的手段提高mtDNA修复能力成为增强细胞对氧化应激耐受性的新策略。外源性上调多种DNA糖基化酶的线粒体表达水平能有效增强mtDNA的修复活性从而提高哺乳动物细胞在氧化应激条件下的存活率。但是由于糖基化酶的底物特异性高,仅能针对单一损伤进行修复,因此更多的研究集中在作用于BER共同通路的修复酶APE1,希望通过增强其活性达到更为广泛的修复增强效应。有研究在线粒体中成功过表达全长野生型APE1基因或APE1的同源基因,但却无显著的细胞效应或产生与预期截然相反的效应。最近有研究报道线粒体型与全长型APE1相比缺乏N端序列却具有比全长型APE1更强的DNA修复活性,然而对于APE1这种缺乏典型的MTS的细胞核编码的基因,其进入线粒体的机制仍未完全明了。因此本研究首先观察在不同的氧化应激刺激下不同细胞株中APE1的亚细胞分布的改变情况,讨论其在氧化应激反应中的生物学效应,然后拟构建N端截短型APE1基因的线粒体表达载体增强其线粒体表达,探讨其对血管内皮细胞mtDNA修复能力的影响,并进一步观察其对细胞在氧化应激状态下凋亡和增殖的影响效应,最后对APE1的线粒体定位信号进行研究,为以线粒体APE1为靶点的基因治疗提供理论基础及可行策略。研究目的 1.探讨不同的处理因素诱导的氧化应激状态下不同细胞中APE1的线粒体转位情况及差异; 2.探讨截短型APE1基因线粒体定位表达对人内皮细胞在氧化应激后存活和增殖能力的影响； 3.探讨APE1线粒体定位信号存在的可能区域，为进一步研究其线粒体定位机制奠定基础。研究内容和方法 1.氧化应激对APE1亚细胞定位的影响：采用激光共聚焦显微镜观察FITC标记的APE1在氧化应激后细胞内定位的变化，同时提取线粒体组分蛋白证实形态学观察。分析氧化应激对APE1亚细胞定位的影响及与不同细胞直接变化的差异，为进一步以线粒体APE1为靶点的基因治疗提供理论基础。 2.截短型APE1线粒体定位表达载体的构建及其生物学效应：以真核表达质粒pcDNA3.1(+)为载体，首先通过PCR得到MTS和ND_APE1序列，而后经过SOE得到拼接的基因，与线性化pcDNA3.1(+)质粒连接后构建pcDNA-mtAPE1-HA，经测序鉴定。采用激光共聚焦显微镜观察转染pcDNA-mtAPE1-HA后的亚细胞定位；Western blot和APE活性分析检测其对HUVE细胞线粒体APE1水平和mtDNA修复能力的增强作用；利用MTT和克隆形成实验检测氧化应激后的细胞存活和增殖能力，用流式细胞仪检测细胞凋亡，并用Western blot检测细胞色素C的胞浆释放。 3. APE1线粒体定位信号的研究：构建C端带有EGFP和HA标签的不同长度的截短型APE1融合蛋白，用激光共聚焦观察细胞内的分布情况推测其线粒体定位信号大致的分布区域。研究结果 1.氧化应激对APE1亚细胞定位的影响：电离辐射后3小时内骨肉瘤细胞中APE1分布由照射前细胞核分布转变为以胞浆为主的分布，3小时线粒体APE1增加超过30倍；H_2O_2处理后3小时内血管内皮HUVE细胞APE1仅少量转位进入线粒体，3小时内线粒体APE1水平增加不超过5倍。 2.截短型APE1线粒体定位表达载体的构建及其生物学效应：经测序分析表明成功构建重组表达载体pcDNA-mtAPE1-HA和对照载体pcDNA-flAPE1-HA。转染HUVE细胞发现pcDNA-mtAPE1-HA能显著增加线粒体APE1水平，而pcDNA-flAPE1-HA则明显增加细胞核APE1水平，两者均能轻度增加胞浆APE1水平。pcDNA-mtAPE1-HA转染能明显增加线粒体APE活性，而不增加胞核APE活性；pcDNA-flAPE1-HA则仅能轻度增加胞核APE活性，不增加线粒体APE活性。在不同浓度H_2O_2诱导的氧化应激后，pcDNA-mtAPE1-HA转染能提高血管内皮细胞细胞存活和增殖能力，同时降低细胞凋亡率，而pcDNA-flAPE1-HA与对照组无显著差异。进一步分析发现，pcDNA-mtAPE1-HA转染可抑制H_2O_2处理后细胞色素C的胞浆释放。 3. APE1线粒体定位信号的研究：带有EGFP标签的载体pEGFP-(42-318)-APE1，pEGFP-(60-318)-APE1，pEGFP-(249-318)-APE1和pEGFP-(288-318)-APE1表达的融合蛋白在细胞内均出现非特异性全细胞弥散分布，而C端带有HA标签的表达载体pEGFP-(42-318)-APE1-HA，pEGFP-(60-318)-APE1-HA和pEGFP-(249-318)-APE1-HA出现特异性线粒体定位而pEGFP-(288-318)-APE1-HA则为非特异性全细胞弥散分布，由于这种亚细胞分布形式差异发生于249-288位氨基酸残基的缺失，因此推测为改区域可能存在线粒体定位信号。结论 1.在电离辐射处理后早期，骨肉瘤HOS细胞内APE1分布由单纯细胞核表达向胞浆为主表达转变；而在H_2O_2处理后早期，血管内皮HUVE细胞内少量APE1由细胞核向线粒体转位。氧化应激后早期APE1线粒体转位是普遍现象，但是其程度和速度与不同细胞株对不同的处理因素敏感性差异相关。 2.通过将线粒体定位序列融合于N端33氨基酸序列缺失的APE1序列并构建真核表达载体转染至血管内皮HUVE细胞中能显著增强线粒体APE1水平，而全长APE1真核表达载体转染仅能增加细胞核APE1水平。 3.截短型APE1线粒体特异性过表达能显著增加血管内皮细胞在H_2O_2诱导的氧化应激后细胞存活及其增殖能力，而全长APE1过表达对H_2O_2诱导的细胞死亡无保护效应。 4.截短型APE1线粒体过表达增加血管内皮细胞在氧化应激后细胞存活，是通过阻断线粒体途径细胞凋亡实现的。 5. APE1的线粒体定位序列可能存在于C端的249-288位氨基酸残基区域内。 6.构建带有蛋白质标签的融合蛋白时，C端较大的标签如EGFP很可能影响APE1线粒体定位序列的暴露，，从而干扰其线粒体定位；而小肽标签如HA则能较好的暴露线粒体定位序列。
[Abstract]:Oxidative stress is closely related with a variety of pathological conditions, including cancer,.DNA aging and cardiovascular disease is one of the important molecular target for oxidative stress and oxidative damage of DNA formed mainly by the base excision repair (BER) repair.APE1 is the rate limiting enzyme in the BER pathway, the expression level of the activity of BER is of paramount importance. Mitochondria is besides nucleus only organelle with genomic DNA, due to the lack of histone protein encoding region dense no intron, coupled with the electron transport chain adjacent, mtDNA is extremely susceptible to oxidative damage, studies have shown that oxidative damage to normal cells steady-state levels of mtDNA is 3-15 times of nDNA. However, the normal cellular mtDNA repair weak activity, through gene therapy method to enhance the mtDNA repair ability has become a new strategy for the enhancement of cellular oxidative stress tolerance. Exogenous upregulation of various DNA glycosylation enzymes of mitochondria The expression level of body can effectively enhance the repair activity of mtDNA so as to improve the survival rate of mammalian cells under oxidative stress conditions. But due to the substrate specificity of glycosylation of the enzyme, can only be repaired for single damage, so more study of APE1 repair enzyme in effect on the BER path, hopes to enhance its the activity reached more extensive repair enhancement. Studies have been successful expression of homologous gene full-length wild-type APE1 gene or APE1 gene in the mitochondria, but no significant effect on cell or produce the opposite effect. Recent studies have reported mitochondrial and full-length APE1 N sequence was compared to the lack of the full length DNA of APE1 repair activity, but for this lack of typical nuclear APE1 encoding MTS gene into the mitochondrial mechanism is still not fully understood. Therefore, this study first To observe the changes of subcellular distribution of APE1 in different cell lines in oxidative stress under different stimulation, discuss its biological effects in response to oxidative stress, and then to construct the N terminal truncated APE1 gene expression vector of enhanced mitochondrial mitochondrial expression, and to investigate its effect on vascular endothelial cell mtDNA repair capacity, and further to observe the effect of apoptosis on oxidative stress and proliferation effect, finally studied the mitochondrial localization signal on APE1, for gene therapy targeting the mitochondrial APE1 and can provide a theoretical basis for the strategy.
research objective
1. to investigate the mitochondrial translocation and difference of APE1 in different cells in different oxidative stress induced by different treatment factors.
2. to investigate the effect of mitochondrial localization and expression of truncated APE1 gene on the survival and proliferation of human endothelial cells after oxidative stress.
3. to explore the possible region of APE1 mitochondrial localization signal, which lays the foundation for further study of the mechanism of mitochondrial localization.
Research contents and methods
1. effects of oxidative stress on the subcellular localization of APE1: the changes observed by APE1 labeled FITC by laser scanning confocal microscope positioning after oxidative stress within the cell, and extract mitochondrial protein components confirmed by morphological observation. Analysis of the effect of oxidative stress on the subcellular localization of APE1 and the differences and different cells directly change, and provide a theoretical basis for further to mitochondrial APE1 target for gene therapy.
2. truncated APE1 mitochondrial targeting expression vector and its biological effect: to eukaryotic expression plasmid pcDNA3.1 (+) as the carrier, first obtained by PCR MTS and ND_APE1 sequence, and then spliced gene obtained by SOE, and linear pcDNA3.1 (+) plasmid was ligated by pcDNA-mtAPE1-HA sequencing. Using confocal laser to observe the subcellular localization of pcDNA-mtAPE1-HA after transfection microscope; detection and analysis of the mitochondrial HUVE cell APE1 level and mtDNA repair capacity enhancement of Western blot and APE activity; the formation of the survival of cells after oxidative stress test and proliferation by MTT and cloning, apoptosis was assayed by flow cytometry, and cytoplasmic Western blot detection the release of cytochrome C.
3., the study of mitochondrial localization signal in APE1: We constructed C truncated APE1 fusion protein with different length of EGFP terminal and HA tag. We observed the distribution of intracellular location by confocal laser scanning and speculated that the location of mitochondrial localization signal was roughly distributed.
Research results
1. effects of oxidative stress on the subcellular localization of APE1: after ionizing radiation of osteosarcoma cells within 3 hours of APE1 distribution by nuclear distribution before irradiation into distribution is mainly located in cytoplasm, 3 hours of mitochondrial APE1 increased more than 30 times within 3 hours after H_2O_2 treatment; vascular endothelial cell APE1 HUVE only a small amount of translocation into mitochondria, mitochondrial APE1 level within 3 hours with no more than 5 times.
2. truncated APE1 mitochondrial targeting expression vector and its biological effect: the sequencing analysis showed that the recombinant expression vector of pcDNA-mtAPE1-HA found that pcDNA-mtAPE1-HA significantly increased the level of mitochondrial APE1 and control vector pcDNA-flAPE1-HA. transfected HUVE cells was successfully constructed, and pcDNA-flAPE1-HA significantly increased the level of nuclear APE1, both can be a slight increase in cytosolic APE1 level.PcDNA-mtAPE1-HA transfection can significantly increase mitochondrial the activity of APE, without increasing nuclear APE activity; pcDNA-flAPE1-HA is only a slight increase in nuclear APE activity, increase the activity of mitochondrial APE in different concentrations. Oxidative stress induced by H_2O_2 after transfection, pcDNA-mtAPE1-HA can improve the survival and proliferation of vascular endothelial cells, and reduce the rate of apoptosis, while the pcDNA-flAPE1-HA and the control group no significant differences. Further analysis found that pcDNA-mtAPE1-HA transfection can inhibit Cytoplasmic release of cytochrome C after H_2O_2 treatment.
Study on 3. APE1 mitochondrial localization signal: vector pEGFP- with EGFP tag (42-318) -APE1, pEGFP- (60-318) -APE1, pEGFP- (249-318) -APE1 and pEGFP- (288-318) expression of -APE1 fusion protein showed nonspecific whole cell dispersed in the cells, and the C terminal with HA tag expression vector pEGFP- (42-318) -APE1-HA, pEGFP- (60-318) -APE1-HA and pEGFP- (249-318) -APE1-HA specific mitochondrial localization and pEGFP- (288-318) -APE1-HA is a non specificity of whole cell dispersion and distribution due to the lack of differences occurred in the form of the subcellular distribution of 249-288 amino acid residues, suggesting that there may be to change the regional mitochondrial localization signal.
conclusion
1. in the early stage after ionizing radiation treatment, the distributions of APE1 in osteosarcoma HOS cells by simple expression in the nucleus to the cytoplasm. The expression changes in H_2O_2 after treatment; early vascular endothelial HUVE cells within a small amount of APE1 from the nucleus to mitochondrial translocation. Early APE1 mitochondrial oxidative stress after the transfer is a common phenomenon, but the extent and speed and different cell lines of different treatment sensitivity.
2., by integrating mitochondrial location sequence into N terminal 33 amino acid sequence deletion APE1 sequence and constructing eukaryotic expression vector, transfection into vascular endothelial HUVE cells can significantly enhance the mitochondrial APE1 level, while full-length APE1 eukaryotic expression vector transfection can only increase the APE1 level of nuclear cells.
3. truncated APE1 mitochondrial specific overexpression can significantly increase the viability and proliferation of vascular endothelial cells after H_2O_2 induced oxidative stress, while full-length APE1 overexpression has no protective effect on H_2O_2 induced cell death.
The overexpression of 4. truncated APE1 mitochondria increases the survival of vascular endothelial cells after oxidative stress, which is achieved by blocking the apoptosis of mitochondria pathway.
The mitochondrial localization sequence of 5. APE1 may exist in the 249-288 - bit amino acid residue region of the C terminal.
6., when constructing protein fusion protein tagged with C, the larger tag at the end of the EGFP, such as APE1, is likely to affect the exposure of APE1 mitochondrial location sequence, thereby interfering with its mitochondrial location. However, small peptide tag, such as HA, can well reveal the mitochondrial localization sequence.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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