發(fā)布時(shí)間：2018-01-07 01:01

  本文關(guān)鍵詞：APE1在線粒體DNA損傷修復(fù)中的作用及其線粒體定位機(jī)制的研究　出處：《第三軍醫(yī)大學(xué)》2008年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 放射治療 骨肉瘤 氧化應(yīng)激 血管內(nèi)皮 基因治療 線粒體 過(guò)表達(dá) DNA損傷修復(fù)基因 脫嘌呤/脫嘧啶核酸內(nèi)切酶 氧化還原因子－1 凋亡 線粒體定位信號(hào)

【摘要】： 氧化應(yīng)激與多種病理狀態(tài)密切相關(guān),包括癌癥,衰老和心血管疾病等。DNA是氧化應(yīng)激損傷的重要靶分子之一,形成的DNA氧化性損傷主要經(jīng)堿基切除修復(fù)(BER)進(jìn)行修復(fù)。APE1為BER途徑的限速酶,其表達(dá)水平高低對(duì)于BER的活性至關(guān)重要。線粒體是除細(xì)胞核外唯一具有基因組DNA的細(xì)胞器,由于缺乏組蛋白,蛋白編碼區(qū)密集無(wú)內(nèi)含子,加之與電子轉(zhuǎn)運(yùn)鏈相鄰,mtDNA極易受到氧化應(yīng)激的損傷,有研究表明正常細(xì)胞mtDNA氧化損傷穩(wěn)態(tài)的水平是nDNA的3-15倍。然而,正常細(xì)胞mtDNA修復(fù)活性較弱,通過(guò)基因治療的手段提高mtDNA修復(fù)能力成為增強(qiáng)細(xì)胞對(duì)氧化應(yīng)激耐受性的新策略。外源性上調(diào)多種DNA糖基化酶的線粒體表達(dá)水平能有效增強(qiáng)mtDNA的修復(fù)活性從而提高哺乳動(dòng)物細(xì)胞在氧化應(yīng)激條件下的存活率。但是由于糖基化酶的底物特異性高,僅能針對(duì)單一損傷進(jìn)行修復(fù),因此更多的研究集中在作用于BER共同通路的修復(fù)酶APE1,希望通過(guò)增強(qiáng)其活性達(dá)到更為廣泛的修復(fù)增強(qiáng)效應(yīng)。有研究在線粒體中成功過(guò)表達(dá)全長(zhǎng)野生型APE1基因或APE1的同源基因,但卻無(wú)顯著的細(xì)胞效應(yīng)或產(chǎn)生與預(yù)期截然相反的效應(yīng)。最近有研究報(bào)道線粒體型與全長(zhǎng)型APE1相比缺乏N端序列卻具有比全長(zhǎng)型APE1更強(qiáng)的DNA修復(fù)活性,然而對(duì)于APE1這種缺乏典型的MTS的細(xì)胞核編碼的基因,其進(jìn)入線粒體的機(jī)制仍未完全明了。因此本研究首先觀察在不同的氧化應(yīng)激刺激下不同細(xì)胞株中APE1的亞細(xì)胞分布的改變情況,討論其在氧化應(yīng)激反應(yīng)中的生物學(xué)效應(yīng),然后擬構(gòu)建N端截短型APE1基因的線粒體表達(dá)載體增強(qiáng)其線粒體表達(dá),探討其對(duì)血管內(nèi)皮細(xì)胞mtDNA修復(fù)能力的影響,并進(jìn)一步觀察其對(duì)細(xì)胞在氧化應(yīng)激狀態(tài)下凋亡和增殖的影響效應(yīng),最后對(duì)APE1的線粒體定位信號(hào)進(jìn)行研究,為以線粒體APE1為靶點(diǎn)的基因治療提供理論基礎(chǔ)及可行策略。 研究目的 1.探討不同的處理因素誘導(dǎo)的氧化應(yīng)激狀態(tài)下不同細(xì)胞中APE1的線粒體轉(zhuǎn)位情況及差異; 2.探討截短型APE1基因線粒體定位表達(dá)對(duì)人內(nèi)皮細(xì)胞在氧化應(yīng)激后存活和增殖能力的影響； 3.探討APE1線粒體定位信號(hào)存在的可能區(qū)域，為進(jìn)一步研究其線粒體定位機(jī)制奠定基礎(chǔ)。 研究?jī)?nèi)容和方法 1.氧化應(yīng)激對(duì)APE1亞細(xì)胞定位的影響：采用激光共聚焦顯微鏡觀察FITC標(biāo)記的APE1在氧化應(yīng)激后細(xì)胞內(nèi)定位的變化，同時(shí)提取線粒體組分蛋白證實(shí)形態(tài)學(xué)觀察。分析氧化應(yīng)激對(duì)APE1亞細(xì)胞定位的影響及與不同細(xì)胞直接變化的差異，為進(jìn)一步以線粒體APE1為靶點(diǎn)的基因治療提供理論基礎(chǔ)。 2.截短型APE1線粒體定位表達(dá)載體的構(gòu)建及其生物學(xué)效應(yīng)：以真核表達(dá)質(zhì)粒pcDNA3.1(+)為載體，首先通過(guò)PCR得到MTS和ND_APE1序列，而后經(jīng)過(guò)SOE得到拼接的基因，與線性化pcDNA3.1(+)質(zhì)粒連接后構(gòu)建pcDNA-mtAPE1-HA，經(jīng)測(cè)序鑒定。采用激光共聚焦顯微鏡觀察轉(zhuǎn)染pcDNA-mtAPE1-HA后的亞細(xì)胞定位；Western blot和APE活性分析檢測(cè)其對(duì)HUVE細(xì)胞線粒體APE1水平和mtDNA修復(fù)能力的增強(qiáng)作用；利用MTT和克隆形成實(shí)驗(yàn)檢測(cè)氧化應(yīng)激后的細(xì)胞存活和增殖能力，用流式細(xì)胞儀檢測(cè)細(xì)胞凋亡，并用Western blot檢測(cè)細(xì)胞色素C的胞漿釋放。 3. APE1線粒體定位信號(hào)的研究：構(gòu)建C端帶有EGFP和HA標(biāo)簽的不同長(zhǎng)度的截短型APE1融合蛋白，用激光共聚焦觀察細(xì)胞內(nèi)的分布情況推測(cè)其線粒體定位信號(hào)大致的分布區(qū)域。 研究結(jié)果 1.氧化應(yīng)激對(duì)APE1亞細(xì)胞定位的影響：電離輻射后3小時(shí)內(nèi)骨肉瘤細(xì)胞中APE1分布由照射前細(xì)胞核分布轉(zhuǎn)變?yōu)橐园麧{為主的分布，3小時(shí)線粒體APE1增加超過(guò)30倍；H_2O_2處理后3小時(shí)內(nèi)血管內(nèi)皮HUVE細(xì)胞APE1僅少量轉(zhuǎn)位進(jìn)入線粒體，3小時(shí)內(nèi)線粒體APE1水平增加不超過(guò)5倍。 2.截短型APE1線粒體定位表達(dá)載體的構(gòu)建及其生物學(xué)效應(yīng)：經(jīng)測(cè)序分析表明成功構(gòu)建重組表達(dá)載體pcDNA-mtAPE1-HA和對(duì)照載體pcDNA-flAPE1-HA。轉(zhuǎn)染HUVE細(xì)胞發(fā)現(xiàn)pcDNA-mtAPE1-HA能顯著增加線粒體APE1水平，而pcDNA-flAPE1-HA則明顯增加細(xì)胞核APE1水平，兩者均能輕度增加胞漿APE1水平。pcDNA-mtAPE1-HA轉(zhuǎn)染能明顯增加線粒體APE活性，而不增加胞核APE活性；pcDNA-flAPE1-HA則僅能輕度增加胞核APE活性，不增加線粒體APE活性。在不同濃度H_2O_2誘導(dǎo)的氧化應(yīng)激后，pcDNA-mtAPE1-HA轉(zhuǎn)染能提高血管內(nèi)皮細(xì)胞細(xì)胞存活和增殖能力，同時(shí)降低細(xì)胞凋亡率，而pcDNA-flAPE1-HA與對(duì)照組無(wú)顯著差異。進(jìn)一步分析發(fā)現(xiàn)，pcDNA-mtAPE1-HA轉(zhuǎn)染可抑制H_2O_2處理后細(xì)胞色素C的胞漿釋放。 3. APE1線粒體定位信號(hào)的研究：帶有EGFP標(biāo)簽的載體pEGFP-(42-318)-APE1，pEGFP-(60-318)-APE1，pEGFP-(249-318)-APE1和pEGFP-(288-318)-APE1表達(dá)的融合蛋白在細(xì)胞內(nèi)均出現(xiàn)非特異性全細(xì)胞彌散分布，而C端帶有HA標(biāo)簽的表達(dá)載體pEGFP-(42-318)-APE1-HA，pEGFP-(60-318)-APE1-HA和pEGFP-(249-318)-APE1-HA出現(xiàn)特異性線粒體定位而pEGFP-(288-318)-APE1-HA則為非特異性全細(xì)胞彌散分布，由于這種亞細(xì)胞分布形式差異發(fā)生于249-288位氨基酸殘基的缺失，因此推測(cè)為改區(qū)域可能存在線粒體定位信號(hào)。 結(jié)論 1.在電離輻射處理后早期，骨肉瘤HOS細(xì)胞內(nèi)APE1分布由單純細(xì)胞核表達(dá)向胞漿為主表達(dá)轉(zhuǎn)變；而在H_2O_2處理后早期，血管內(nèi)皮HUVE細(xì)胞內(nèi)少量APE1由細(xì)胞核向線粒體轉(zhuǎn)位。氧化應(yīng)激后早期APE1線粒體轉(zhuǎn)位是普遍現(xiàn)象，但是其程度和速度與不同細(xì)胞株對(duì)不同的處理因素敏感性差異相關(guān)。 2.通過(guò)將線粒體定位序列融合于N端33氨基酸序列缺失的APE1序列并構(gòu)建真核表達(dá)載體轉(zhuǎn)染至血管內(nèi)皮HUVE細(xì)胞中能顯著增強(qiáng)線粒體APE1水平，而全長(zhǎng)APE1真核表達(dá)載體轉(zhuǎn)染僅能增加細(xì)胞核APE1水平。 3.截短型APE1線粒體特異性過(guò)表達(dá)能顯著增加血管內(nèi)皮細(xì)胞在H_2O_2誘導(dǎo)的氧化應(yīng)激后細(xì)胞存活及其增殖能力，而全長(zhǎng)APE1過(guò)表達(dá)對(duì)H_2O_2誘導(dǎo)的細(xì)胞死亡無(wú)保護(hù)效應(yīng)。 4.截短型APE1線粒體過(guò)表達(dá)增加血管內(nèi)皮細(xì)胞在氧化應(yīng)激后細(xì)胞存活，是通過(guò)阻斷線粒體途徑細(xì)胞凋亡實(shí)現(xiàn)的。 5. APE1的線粒體定位序列可能存在于C端的249-288位氨基酸殘基區(qū)域內(nèi)。 6.構(gòu)建帶有蛋白質(zhì)標(biāo)簽的融合蛋白時(shí)，C端較大的標(biāo)簽如EGFP很可能影響APE1線粒體定位序列的暴露，，從而干擾其線粒體定位；而小肽標(biāo)簽如HA則能較好的暴露線粒體定位序列。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R363

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相關(guān)期刊論文 前2條

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2 卿毅;王東;仲召陽(yáng);李增鵬;張沁宏;;pSilence APE1提高骨肉瘤放療敏感性的動(dòng)物實(shí)驗(yàn)研究[J];中國(guó)腫瘤臨床;2007年04期

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