本文關(guān)鍵詞：DNA復(fù)制壓力和活性氧誘發(fā)γ-H2AX陽(yáng)性微核及其機(jī)制研究　出處：《山東大學(xué)》2012年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 微核 H2AX磷酸化 γ-H2AX陽(yáng)性微核 DNA復(fù)制壓力 活性氧

【摘要】：微核是指位于細(xì)胞漿中,獨(dú)立于細(xì)胞主核之外的圓形或者橢圓形的微小核。傳統(tǒng)觀點(diǎn)認(rèn)為微核的形成原因是細(xì)胞的染色體受到損傷發(fā)生斷裂或者牽引染色體的紡錘絲出現(xiàn)故障,導(dǎo)致細(xì)胞在進(jìn)入下一次有絲分裂時(shí),染色體片段或者整條染色體不能隨有絲分裂進(jìn)入子細(xì)胞,即微核主要形成于有絲分裂后期。但是目前有多篇報(bào)道稱(chēng)由于染色體重塑異常、癌基因擴(kuò)增等原因可以導(dǎo)致細(xì)胞在分裂間期形成微核。微核作為一種重要的遺傳毒理學(xué)和基因組不穩(wěn)定性的指標(biāo),可用于指示遺傳物質(zhì)的損傷程度�，F(xiàn)在國(guó)內(nèi)外常通過(guò)檢測(cè)外周血淋巴細(xì)胞以及口腔、鼻腔等部位剝落的上皮細(xì)胞的微核發(fā)生頻率來(lái)評(píng)價(jià)各種傷害引起的細(xì)胞遺傳學(xué)損傷程度,由此可見(jiàn)微核分析的普遍性和重要性。但是,作為一種如此重要的基因組不穩(wěn)定性的監(jiān)測(cè)指標(biāo),微核的發(fā)生機(jī)制還沒(méi)有完全的定論。 除微核以外,組蛋白H2AX的磷酸化,簡(jiǎn)稱(chēng)γ-H2AX,也是判斷DNA損傷的重要指標(biāo)。一旦DNA發(fā)生雙鏈斷裂損傷,就可以激活A(yù)TM/ATR通路,使DNA雙鏈斷裂部位的組蛋白H2AX在絲氨酸139位迅速的發(fā)生磷酸化。組蛋白H2AX發(fā)生磷酸化后,大量的DNA修復(fù)因子被招募到損傷位點(diǎn)處,并對(duì)損傷進(jìn)行修復(fù)。使用γ-H2AX的抗體進(jìn)行免疫熒光實(shí)驗(yàn)可以檢測(cè)到H2AX的磷酸化,并在熒光顯微鏡下觀察到可見(jiàn)的γ-H2AX染色信號(hào),這已經(jīng)成為測(cè)定DNA雙鏈斷裂損傷的金標(biāo)準(zhǔn)。γ-H2AX染色信號(hào)主要以均勻彌散的γ-H2AX染色以及γ-H2AX焦點(diǎn)兩種形式存在。當(dāng)細(xì)胞遭遇DNA復(fù)制壓力、受到高濃度的氯化鈉處理、或者經(jīng)過(guò)乏氧處理后,細(xì)胞核中會(huì)出現(xiàn)均勻彌散的γ-H2AX染色；而細(xì)胞受到電離輻射處理后,細(xì)胞核中會(huì)出現(xiàn)大量分散的γ-H2AX焦點(diǎn)。因此,對(duì)受到損傷因素處理的細(xì)胞而言,通過(guò)觀察其H2AX磷酸化的程度和分布形式,可以幫助我們對(duì)其DNA雙鏈斷裂損傷進(jìn)行定位及定量的研究。 活性氧(ROS)是生物體內(nèi)產(chǎn)生的超氧陰離子、過(guò)氧化氫、羥自由基、一氧化氮等活性含氧化合物的總稱(chēng)。很多內(nèi)源性因素以及外源性因素都能夠?qū)е翿OS的產(chǎn)生。如果細(xì)胞不能維持內(nèi)環(huán)境中ROS水平的穩(wěn)定,則會(huì)導(dǎo)致一系列嚴(yán)重后果。因此,為了維持正常的生命活動(dòng),細(xì)胞通過(guò)多種機(jī)制對(duì)ROS水平進(jìn)行調(diào)控,從而維持內(nèi)環(huán)境的穩(wěn)定,使細(xì)胞維持正常的生長(zhǎng)和代謝。ROS與DNA損傷之間存在密切關(guān)系。一方面,ROS能夠?qū)е露喾N形式的DNA損傷,如DNA構(gòu)象改變、堿基損傷、單鏈斷裂損傷以及雙鏈斷裂損傷。另一方面,持續(xù)的DNA損傷能夠?qū)е翿OS水平增高。越來(lái)越多的研究表明,DNA修復(fù)的關(guān)鍵蛋白丟失后,能夠造成嚴(yán)重的DNA損傷,從而導(dǎo)致ROS水平的增高。綜上所述,ROS可導(dǎo)致多種形式的DNA損傷,后者也可以反過(guò)來(lái)導(dǎo)致ROS水平的增高,兩者之間存在正反饋?zhàn)饔谩?我們?cè)趯?duì)多種哺乳動(dòng)物細(xì)胞系進(jìn)行γ-H2AX免疫熒光實(shí)驗(yàn)的過(guò)程中觀察到某些微核呈現(xiàn)均勻的彌散性的γ-H2AX染色,稱(chēng)之為γ-H2AX陽(yáng)性微核或MN-γ-H2AX(+)。為了進(jìn)一步明確這類(lèi)新型微核的存在及其特征,并深入探討這類(lèi)新型微核的發(fā)生機(jī)制,本課題從以下兩方面進(jìn)行研究： 第一部分 DNA復(fù)制壓力誘發(fā)含有大量DNA雙鏈斷裂損傷的新型MN-γ-H2AX(+) 為了明確MN-γ-H2AX(+)這類(lèi)新型微核發(fā)生的普遍性,我們對(duì)多種哺乳動(dòng)物細(xì)胞進(jìn)行免疫熒光實(shí)驗(yàn),并選擇了人類(lèi)乳腺癌細(xì)胞系MCF-7進(jìn)行進(jìn)一步的研究。通過(guò)使用藥物阻止DNA復(fù)制或使用具有DNA復(fù)制遺傳缺陷的細(xì)胞進(jìn)行實(shí)驗(yàn),我們分析了DNA復(fù)制壓力與這類(lèi)新型微核形成的關(guān)系。實(shí)驗(yàn)結(jié)果如下： 1.通過(guò)對(duì)多種哺乳動(dòng)物細(xì)胞進(jìn)行免疫熒光實(shí)驗(yàn)和微核計(jì)數(shù),結(jié)果發(fā)現(xiàn)： 1)微核分為MN-γ-H2AX(+)和MN-γ-H2AX(-)。MN-γ-H2AX(+)的特征為：微核中顯示出均勻彌散的γ-H2AX染色,并且這種彌散性的γ-H2AX染色幾乎占據(jù)了整個(gè)微核。 2)MN-γ-H2AX(+)的存在具有普遍性。不同細(xì)胞中MN-γ-H2AX(+)的發(fā)生頻率有所不同,大約在0.5%到4%之間,占總微核的比例數(shù)在20%-50%之間。 2.使用藥物處理MCF-7細(xì)胞,統(tǒng)計(jì)MN-γ-H2AX(+)的發(fā)生頻率,結(jié)果如下：1)使用流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期分布情況。結(jié)果顯示,未經(jīng)藥物處理組的S期細(xì)胞比例數(shù)為30%左右,而羥基脲、胸苷嘧啶、阿非迪霉素處理組的S期細(xì)胞比例數(shù)增至60%左右,說(shuō)明藥物處理使細(xì)胞停滯于S期。 2)使用導(dǎo)致S期停滯的藥物羥基脲、阿非迪霉素、胸苷嘧啶處理MCF-7細(xì)胞,微核計(jì)數(shù)結(jié)果顯示MN-γ-H2AX(+)發(fā)生頻率的增加幅度遠(yuǎn)大于MN-γ-H2AX(-)發(fā)生頻率的增加幅度。 3)紫杉醇通過(guò)抑制微管解聚使細(xì)胞停滯于有絲分裂中期,使用紫杉醇處理MCF-7細(xì)胞,產(chǎn)生的主要是MN-γ-H2AX(-),而MN-γ-H2AX(+)的發(fā)生頻率并沒(méi)有明顯改變。 3.使用不同藥物(阿非迪霉素、羥基脲、胸苷嘧啶、紫杉醇)處理MCF-7細(xì)胞24h后,更換為新鮮完全培養(yǎng)基釋放不同時(shí)間(0h,6h,24h,48h,72h,96h),微核計(jì)數(shù)結(jié)果顯示： 1)藥物處理后未經(jīng)釋放的細(xì)胞中,MN-γ-H2AX(+)的發(fā)生頻率已有明顯增高；藥物處理后分別進(jìn)行6h.24h.48h.72h釋放的細(xì)胞中,MN-γ-H2AX(+)的發(fā)生頻率均高于未經(jīng)藥物處理的對(duì)照組細(xì)胞；藥物處理后進(jìn)行96h釋放的細(xì)胞中,MN-γ-H2AX(+)的發(fā)生頻率幾乎恢復(fù)到本底水平。 2)根據(jù)藥物處理后,細(xì)胞核中γ-H2AX染色的情況將細(xì)胞分為三類(lèi)：對(duì)于整個(gè)細(xì)胞核均呈現(xiàn)出均勻彌散性γ-H2AX染色的細(xì)胞,我們將其定義為一類(lèi)細(xì)胞；對(duì)于細(xì)胞核中存在大量γ-H2AX焦點(diǎn)的細(xì)胞,我們將其定義為二類(lèi)細(xì)胞；對(duì)于細(xì)胞核中幾乎沒(méi)有γ-H2AX信號(hào)的細(xì)胞,我們將其定義為三類(lèi)細(xì)胞。結(jié)合各類(lèi)細(xì)胞占總細(xì)胞的比例以及細(xì)胞周期結(jié)果推測(cè),藥物處理后所檢測(cè)到的一類(lèi)細(xì)胞與二類(lèi)細(xì)胞很有可能是處于S期且經(jīng)歷DNA復(fù)制壓力的細(xì)胞。藥物處理后對(duì)不同釋放時(shí)間各類(lèi)細(xì)胞產(chǎn)生的微核數(shù)進(jìn)行統(tǒng)計(jì),結(jié)果發(fā)現(xiàn),使用阻止DNA復(fù)制的藥物處理后,未經(jīng)釋放的情況下,大多數(shù)新產(chǎn)生的MN-γ-H2AX(+)都是由處于S期的一類(lèi)細(xì)胞與二類(lèi)細(xì)胞產(chǎn)生的。由此明確了MN-γ-H2AX(+)是在S期形成的。 4.免疫熒光實(shí)驗(yàn)觀察小鼠皮膚成纖維細(xì)胞(MSF)發(fā)現(xiàn)有γ-H2AX信號(hào)在細(xì)胞核外周聚集成簇并且有向外突出的趨勢(shì),推測(cè)這樣的細(xì)胞核突出部位很可能是MN-γ-H2AX(+)的前體。 5.使用具有DNA復(fù)制遺傳缺陷的細(xì)胞進(jìn)行實(shí)驗(yàn),結(jié)果發(fā)現(xiàn)： 1)通過(guò)流式細(xì)胞術(shù)和BrdU摻入實(shí)驗(yàn)證明,在RPA1低表達(dá)的MCF-7細(xì)胞中存在DNA復(fù)制壓力。 2)通過(guò)微核計(jì)數(shù)發(fā)現(xiàn),與對(duì)照細(xì)胞相比,RPA1(?)(?)表達(dá)細(xì)胞中MN-γ-H2AX(+)的發(fā)生頻率有所增高。 綜上所述,MN-γ-H2AX(+)是一種新型微核,它不同于傳統(tǒng)意義上有絲分裂后期形成的微核,主要是在細(xì)胞分裂間期形成的。我們推測(cè)這類(lèi)微核的形成原因是細(xì)胞在S期遭遇DNA復(fù)制壓力,進(jìn)而導(dǎo)致了大量的DNA雙鏈斷裂損傷,這些無(wú)法修復(fù)的損傷部位聚集在一起排出細(xì)胞主核,形成MN-γ-H2AX(+)。研究這類(lèi)新型的MN-γ-H2AX(+)有助于我們認(rèn)識(shí)DNA復(fù)制壓力所引起的基因組不穩(wěn)定性,以及評(píng)估導(dǎo)致DNA復(fù)制壓力的遺傳和環(huán)境因素。 第二部分 活性氧(ROS)導(dǎo)致MN-γ-H2AX(+)發(fā)生頻率增高 第一部分的研究證明DNA復(fù)制壓力能夠誘發(fā)MN-γ-H2AX(+),且有多項(xiàng)研究證明ROS能夠?qū)е虏煌问降腄NA損傷,因此我們接下來(lái)研究了ROS水平與MN-γ-H2AX(+)形成之間的關(guān)系。我們使用藥物影響ROS水平以及使用具有抗氧化基因缺陷的細(xì)胞進(jìn)行實(shí)驗(yàn),檢測(cè)MN-γ-H2AX(+)的發(fā)生頻率；同時(shí)使用具有抗氧化作用的藥物對(duì)以上細(xì)胞進(jìn)行聯(lián)合處理,以抑制細(xì)胞內(nèi)ROS水平的增高,分析ROS與MN-γ-H2AX(+)形成的關(guān)系。 1.使用導(dǎo)致ROS水平增高的藥物過(guò)氧化氫(H202)以及具有抗氧化作用的藥物N-乙酰半胱氨酸(NAC)處理細(xì)胞,結(jié)果如下： 1)使用不同濃度的H2O2(50μM,100μM,150μM)處理U20S細(xì)胞不同時(shí)間(2h,6h,12h,24h),隨后更換為新鮮完全培養(yǎng)基釋放48h,檢測(cè)MN-γ-H2AX(+)的形成情況。結(jié)果顯示,H202能夠誘發(fā)MN-γ-H2AX(+),且呈劑量依賴(lài)性和時(shí)間依賴(lài)性。 2)使用具有抗氧化作用的藥物NAC與H202聯(lián)合處理細(xì)胞,結(jié)果發(fā)現(xiàn),NAC能夠下調(diào)H202所造成的MN-γ-H2AX(+)發(fā)生頻率的增高。從而通過(guò)藥物實(shí)驗(yàn)證明了ROS能夠誘發(fā)MN-γ-H2AX(+)。 2.通過(guò)不同方式使具有抗氧化作用的基因p53功能改變后,檢測(cè)MN-γ-H2AX(+)發(fā)生頻率的變化情況。結(jié)果如下： 1)使用p53激活劑nutlin-3處理U20S細(xì)胞,MN-γ-H2AX(+)的發(fā)生頻率有所降低。 2)使用p53抑制劑pifithrin-α處理U20S細(xì)胞,MN-γ-H2AX(+)的發(fā)生頻率有明顯增高。 3)使用p53功能缺陷的U20S細(xì)胞以及p53缺失的小鼠皮膚成纖維細(xì)胞進(jìn)行實(shí)驗(yàn),結(jié)果發(fā)現(xiàn)：與對(duì)照細(xì)胞相比,p53功能缺陷的細(xì)胞中,MN-γ-H2AX(+)的發(fā)生頻率有明顯增高。 4)使用具有抗氧化作用的藥物NAC處理p53功能缺陷細(xì)胞及其對(duì)照細(xì)胞,結(jié)果發(fā)現(xiàn)：NAC能夠下調(diào)由于p53功能缺陷而導(dǎo)致的MN-γ-H2AX(+)發(fā)生頻率的增高。 3.為了了解p53下游基因?qū)N-γ-H2AX(+)形成的影響,我們使用siRNA抑制p53下游具有抗氧化作用的基因SESNl的表達(dá)。結(jié)果發(fā)現(xiàn),與對(duì)照細(xì)胞相比,SESN1低表達(dá)的細(xì)胞中,MN-γ-H2AX(+)的發(fā)生頻率有所增高。 4.有文獻(xiàn)報(bào)道p400受到抑制后,細(xì)胞內(nèi)ROS水平有所增高。因此,我們使用p400低表達(dá)的U20S細(xì)胞進(jìn)行實(shí)驗(yàn),結(jié)果發(fā)現(xiàn)p400表達(dá)量降低后,MN-γ-H2AX(+)的發(fā)生頻率明顯增高。進(jìn)一步證明了ROS能夠誘發(fā)MN-γ-H2AX(+)。 綜上所述,ROS水平增高能夠誘發(fā)MN-γ-H2AX(+)。
[Abstract]:The micronucleus is located in cytoplasm, nuclear small independent cells nucleus round or oval. The traditional view is that the reason is the formation of micronucleus cell chromosome by spindle injury or fracture traction chromosome failure, resulting in a cell into mitosis, chromosome fragments or whole chromosomes not with the entry into mitosis cells, micronucleus formed mainly in late mitosis. But there are a number of reports due to abnormal chromosome remodeling, causes cancer gene amplification can lead to cell micronucleus formation in interphase micronucleus. As a kind of important genetic toxicology and genomic instability index, can be used for damage indicating the genetic material. Now both at home and abroad through the detection of peripheral blood lymphocytes and oral cavity, peeling and other parts of the epithelial cell micronucleus. Frequency can be used to evaluate the degree of cytogenetic damage caused by various injuries, which shows the universality and importance of micronucleus analysis. However, as a monitoring index for such an important genomic instability, the mechanism of micronucleus has not yet been completely conclusive.
In addition to the micronucleus, the phosphorylation of histone H2AX, referred to as -H2AX gamma, is also an important index to judge the damage of DNA. Once the double strand breaks in DNA, you can activate the ATM/ATR pathway, the DNA double strand break sites of histone H2AX at serine 139 phosphorylation. The rapid phosphorylation of histone H2AX after a large number of DNA repair factors are recruited to the injury site, and to repair the damage. The use of gamma -H2AX antibody by immunofluorescence test can detect the phosphorylation of H2AX, and observed under the fluorescence microscope gamma -H2AX visible staining signal, which has become the gold standard of damage was determined by DNA double strand breaks. Gamma -H2AX staining signals mainly homogeneously staining and gamma gamma -H2AX -H2AX focus two forms exist. When cells encounter DNA replication stress, by treatment of high concentration sodium chloride, or after hypoxia treatment, will be out of the nucleus The uniform dispersion of gamma -H2AX staining; and the cells were treated after ionizing radiation, the emergence of a large number of scattered gamma -H2AX will focus on the nucleus. Therefore, the treatment of the injury by cell factors, the degree and distribution of observed H2AX phosphorylation, we can study the damage localization and quantitation of DNA double strand breaks help.
Reactive oxygen species (ROS), superoxide anion, hydrogen peroxide in the organism, hydroxyl radical, general nitric oxide and other reactive oxygen compounds. Many endogenous factors and exogenous factors can lead to ROS. If the level of ROS cells are unable to maintain the internal environment of stability, it will lead to a series of serious consequences. Therefore, in order to maintain normal life activities of cells to regulate the level of ROS through a variety of mechanisms, so as to maintain the stability of the environment, to maintain the growth and metabolism of.ROS cells between normal and DNA damage are closely related. On the one hand, ROS can cause DNA damage in various forms, such as changing the conformation of DNA, base damage, single strand breaks and double strand breaks. On the other hand, persistent DNA damage can lead to elevated levels of ROS. More and more studies show that the key protein of DNA repair after the loss, can cause serious Heavy DNA damage leads to the increase of ROS level. In conclusion, ROS can lead to various forms of DNA damage, which in turn can lead to the increase of ROS level, and there is a positive feedback between them.
We are in the process of gamma -H2AX immunofluorescence experiments on a variety of mammalian cell lines observed in some micronuclei showed homogeneous diffuse gamma -H2AX staining, called -H2AX or MN- gamma gamma positive micronucleus -H2AX (+). In order to further clarify the existence and characteristics of this new type of micronucleus, and in-depth study of the mechanism of this kind of new type of micronuclei, this study was carried out from the following two aspects:
Part one
DNA replication pressure induces a new type of MN- gamma -H2AX (+) containing a large number of DNA double strand breaks
In order to clarify the MN- gamma -H2AX (+) universality of this new type of micronucleus, we performed immunofluorescence experiments on mammalian cells, and selection of human breast cancer cell line MCF-7 were further studied. Through the use of drugs to prevent DNA replication or use a DNA copy of genetic defects of cell experiment, we analyzed DNA copy of this kind of relationship between pressure and micronucleus formation. The experimental results are as follows:
1. by immunofluorescence experiments and micronucleus counts of many mammalian cells, the results are as follows:
1) micronucleus is divided into MN- gamma -H2AX (+) and MN- gamma -H2AX (-).MN- gamma -H2AX (+). The characteristics of micronucleus are: uniformly dispersed gamma -H2AX staining in micronucleus, and the diffuse gamma -H2AX staining almost occupies the whole micronucleus.
2) the existence of MN- gamma -H2AX (+) is universal. The occurrence frequency of MN- gamma -H2AX (+) in different cells is different, about 0.5% to 4%, and the proportion of total micronucleus is between 20%-50%.
2. the use of drug treatment of MCF-7 cells, MN- y -H2AX (+) the occurrence frequency, the results are as follows: 1) using flow cytometry to detect the cell cycle distribution. The results show that without drug treatment group the proportion of cells in S phase was about 30% and the number of hydroxyurea, and thymidine, A Fei Di kanamycin treatment the proportion of S phase cell number increased to about 60%, indicating that the drug treatment of cells in the S phase of stagnation.
2) to S arrest drug hydroxyurea, aphidicolin, thymidine MCF-7 cells, micronucleus counting results showed MN- (+) -H2AX gamma frequency increase is far greater than the MN- gamma -H2AX (-) frequency increases.
3) paclitaxel blocked cells in the middle phase of mitosis by inhibiting microtubule depolymerization. The MN- gamma -H2AX (-) was mainly produced by paclitaxel treatment of MCF-7 cells, while the frequency of MN- gamma -H2AX (+) did not change significantly.
3. the use of different drugs (aphidicolin, hydroxyurea, thymidine, paclitaxel) treatment of MCF-7 cells after 24h, the replacement for the fresh complete medium release at different time (0h, 6h, 24h, 48h, 72h, 96h), micronucleus counts showed:
1) after treatment without the release of cells, MN- y -H2AX (+) the occurrence frequency has been significantly increased after treatment respectively; the release of 6h.24h.48h.72h cells, MN- y -H2AX (+) the occurrence frequency were higher than control group cells by drugs; drug treatment after the release of 96h cells in MN-, gamma -H2AX (+) the frequency is almost back to the background level.
2) according to the drug treatment, the nucleus gamma -H2AX the staining of the cells were divided into three categories: the nucleus showed homogeneous diffuse gamma -H2AX staining cells, we define it as a kind of cell; for the existence of a large number of -H2AX cell nuclei in the gamma focus, we define it as the two class the nuclei of cells; almost no gamma signal in -H2AX cells, we define the three kinds of cells. The combination of all kinds of cells in the total cell and the ratio of cell cycle results suggested that a class of drug treated cells after detected with two kinds of cells is likely to be in phase S and DNA pressure cell replication experience the number of micronuclei. For different release time of all kinds of cells after treatment of the statistical results showed that the use of stop DNA replication after drug treatment, without the release of the case, most of the new generation of MN- gamma -H2AX (+) are A class of cells in the S phase and two types of cells are produced. Thus, it is clear that MN- gamma -H2AX (+) is formed during the S phase.
4. immunofluorescence assay was used to observe the skin fibroblasts (MSF) in mice. It was found that the -H2AX MN- signal was clustered in the periphery of the nucleus and showed a trend of outward protrusion. It is presumed that such a nucleus prominent part is the precursor of MN- gamma -H2AX +.
5. the experiment was conducted using a cell with DNA replicating genetic defects, and the results were as follows:
1) through flow cytometry and BrdU incorporation experiments, it was proved that there was DNA replication pressure in the low expression of RPA1 MCF-7 cells.
2) by micronucleus count, the frequency of MN- gamma -H2AX (+) in the RPA1 (?) (?) expression cells was higher than that of the control cells.
In summary, MN- y -H2AX (+) is a new type of micronucleus, it is different from the traditional sense of the late mitosis formation of micronuclei in interphase cells, is the main form. We speculate that the reason for this is the kind of micronucleus cells in S phase with DNA replication stress, leading to a large number of DNA double strand breaks these injuries, can not repair the injury site together out of the cell nucleus, the formation of MN- gamma -H2AX (+). This kind of new MN- gamma -H2AX (+) contributes to our understanding of DNA replication stress caused by genomic instability, and the evaluation resulted in DNA replication stress of genetic and environmental factors.
The second part
Active oxygen (ROS) leads to an increase in the frequency of MN- gamma -H2AX (+)
The first part of the study demonstrated that DNA replication stress induces MN- gamma -H2AX (+), and there are a number of studies have proved that ROS can cause DNA damage in different forms, so we next studied the ROS level and MN- y -H2AX (+). We use the relationship between drugs affect the level of ROS and the use of antioxidant deficient cells experiments were conducted to detect MN- gamma -H2AX (+) the frequency of drug use; at the same time the antioxidant combined treatment on the above cells, to inhibit the increase of intracellular ROS levels, analysis of ROS and MN- gamma -H2AX (+) to form a relationship.
1. the use of drug hydrogen peroxide (H202), which leads to a higher level of ROS, and the antioxidation drug N- acetylcysteine (NAC), can be used to treat cells. The results are as follows:
1) with different concentrations of H2O2 (50 M, 100 M, 150 M) with different time of U20S cells (2h, 6h, 12h, 24h), then replaced with fresh complete medium release 48h, detection of MN- gamma -H2AX (+) formation. The results showed that H202 can induce MN- gamma -H2AX (+), and there was a dose and time dependent manner.
2) using antioxidant drugs NAC and H202 to treat cells, it is found that NAC can down regulate the frequency of MN- gamma -H2AX (+) induced by H202, which proves that ROS can induce MN- -H2AX (+) through drug experiments.
2. the changes in the frequency of MN- gamma -H2AX (+) were detected by changing the function of the antioxidant p53 gene in different ways. The results were as follows:
1) the frequency of MN- gamma -H2AX (+) was reduced by the use of p53 activator nutlin-3 to treat U20S cells.
2) the frequency of MN- gamma -H2AX (+) was significantly increased by the use of p53 inhibitor pifithrin- alpha to treat U20S cells.
3) using p53 deficient U20S cells and p53 deficient mouse skin fibroblasts, we found that compared with control cells, the frequency of MN- gamma -H2AX (+) increased significantly in p53 deficient cells.
4) using NAC, an antioxidant drug, to treat p53 defective cells and their control cells, it is found that NAC can reduce the frequency of MN- gamma -H2AX (+) caused by p53 dysfunction.
3., in order to understand the effect of p53 downstream gene on the formation of MN- gamma -H2AX (+), we used siRNA to inhibit the expression of SESNl gene with antioxidant effect downstream of p53. It was found that the frequency of MN- -H2AX (+) increased in cells with low SESN1 expression compared with control cells.
4., it has been reported that the level of ROS is increased after P400 is inhibited. Therefore, we used P400 low expression U20S cells to carry out the experiment. It was found that the frequency of MN- -H2AX (+) increased significantly after the decrease of P400 expression. It further proved ROS could induce MN- gamma -H2AX (+).
To sum up, the increased level of ROS can induce MN- gamma -H2AX (+).

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：Q343;R394.3

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