DNA复制压力和活性氧诱发γ-H2AX阳性微核及其机制研究

发布时间：2018-01-01 20:37

本文关键词：DNA复制压力和活性氧诱发γ-H2AX阳性微核及其机制研究　出处：《山东大学》2012年博士论文　论文类型：学位论文

【摘要】：微核是指位于细胞浆中,独立于细胞主核之外的圆形或者椭圆形的微小核。传统观点认为微核的形成原因是细胞的染色体受到损伤发生断裂或者牵引染色体的纺锤丝出现故障,导致细胞在进入下一次有丝分裂时,染色体片段或者整条染色体不能随有丝分裂进入子细胞,即微核主要形成于有丝分裂后期。但是目前有多篇报道称由于染色体重塑异常、癌基因扩增等原因可以导致细胞在分裂间期形成微核。微核作为一种重要的遗传毒理学和基因组不稳定性的指标,可用于指示遗传物质的损伤程度。现在国内外常通过检测外周血淋巴细胞以及口腔、鼻腔等部位剥落的上皮细胞的微核发生频率来评价各种伤害引起的细胞遗传学损伤程度,由此可见微核分析的普遍性和重要性。但是,作为一种如此重要的基因组不稳定性的监测指标,微核的发生机制还没有完全的定论。除微核以外,组蛋白H2AX的磷酸化,简称γ-H2AX,也是判断DNA损伤的重要指标。一旦DNA发生双链断裂损伤,就可以激活ATM/ATR通路,使DNA双链断裂部位的组蛋白H2AX在丝氨酸139位迅速的发生磷酸化。组蛋白H2AX发生磷酸化后,大量的DNA修复因子被招募到损伤位点处,并对损伤进行修复。使用γ-H2AX的抗体进行免疫荧光实验可以检测到H2AX的磷酸化,并在荧光显微镜下观察到可见的γ-H2AX染色信号,这已经成为测定DNA双链断裂损伤的金标准。γ-H2AX染色信号主要以均匀弥散的γ-H2AX染色以及γ-H2AX焦点两种形式存在。当细胞遭遇DNA复制压力、受到高浓度的氯化钠处理、或者经过乏氧处理后,细胞核中会出现均匀弥散的γ-H2AX染色；而细胞受到电离辐射处理后,细胞核中会出现大量分散的γ-H2AX焦点。因此,对受到损伤因素处理的细胞而言,通过观察其H2AX磷酸化的程度和分布形式,可以帮助我们对其DNA双链断裂损伤进行定位及定量的研究。活性氧(ROS)是生物体内产生的超氧阴离子、过氧化氢、羟自由基、一氧化氮等活性含氧化合物的总称。很多内源性因素以及外源性因素都能够导致ROS的产生。如果细胞不能维持内环境中ROS水平的稳定,则会导致一系列严重后果。因此,为了维持正常的生命活动,细胞通过多种机制对ROS水平进行调控,从而维持内环境的稳定,使细胞维持正常的生长和代谢。ROS与DNA损伤之间存在密切关系。一方面,ROS能够导致多种形式的DNA损伤,如DNA构象改变、碱基损伤、单链断裂损伤以及双链断裂损伤。另一方面,持续的DNA损伤能够导致ROS水平增高。越来越多的研究表明,DNA修复的关键蛋白丢失后,能够造成严重的DNA损伤,从而导致ROS水平的增高。综上所述,ROS可导致多种形式的DNA损伤,后者也可以反过来导致ROS水平的增高,两者之间存在正反馈作用。我们在对多种哺乳动物细胞系进行γ-H2AX免疫荧光实验的过程中观察到某些微核呈现均匀的弥散性的γ-H2AX染色,称之为γ-H2AX阳性微核或MN-γ-H2AX(+)。为了进一步明确这类新型微核的存在及其特征,并深入探讨这类新型微核的发生机制,本课题从以下两方面进行研究：第一部分 DNA复制压力诱发含有大量DNA双链断裂损伤的新型MN-γ-H2AX(+) 为了明确MN-γ-H2AX(+)这类新型微核发生的普遍性,我们对多种哺乳动物细胞进行免疫荧光实验,并选择了人类乳腺癌细胞系MCF-7进行进一步的研究。通过使用药物阻止DNA复制或使用具有DNA复制遗传缺陷的细胞进行实验,我们分析了DNA复制压力与这类新型微核形成的关系。实验结果如下： 1.通过对多种哺乳动物细胞进行免疫荧光实验和微核计数,结果发现： 1)微核分为MN-γ-H2AX(+)和MN-γ-H2AX(-)。MN-γ-H2AX(+)的特征为：微核中显示出均匀弥散的γ-H2AX染色,并且这种弥散性的γ-H2AX染色几乎占据了整个微核。 2)MN-γ-H2AX(+)的存在具有普遍性。不同细胞中MN-γ-H2AX(+)的发生频率有所不同,大约在0.5%到4%之间,占总微核的比例数在20%-50%之间。 2.使用药物处理MCF-7细胞,统计MN-γ-H2AX(+)的发生频率,结果如下：1)使用流式细胞术检测细胞周期分布情况。结果显示,未经药物处理组的S期细胞比例数为30%左右,而羟基脲、胸苷嘧啶、阿非迪霉素处理组的S期细胞比例数增至60%左右,说明药物处理使细胞停滞于S期。 2)使用导致S期停滞的药物羟基脲、阿非迪霉素、胸苷嘧啶处理MCF-7细胞,微核计数结果显示MN-γ-H2AX(+)发生频率的增加幅度远大于MN-γ-H2AX(-)发生频率的增加幅度。 3)紫杉醇通过抑制微管解聚使细胞停滞于有丝分裂中期,使用紫杉醇处理MCF-7细胞,产生的主要是MN-γ-H2AX(-),而MN-γ-H2AX(+)的发生频率并没有明显改变。 3.使用不同药物(阿非迪霉素、羟基脲、胸苷嘧啶、紫杉醇)处理MCF-7细胞24h后,更换为新鲜完全培养基释放不同时间(0h,6h,24h,48h,72h,96h),微核计数结果显示： 1)药物处理后未经释放的细胞中,MN-γ-H2AX(+)的发生频率已有明显增高；药物处理后分别进行6h.24h.48h.72h释放的细胞中,MN-γ-H2AX(+)的发生频率均高于未经药物处理的对照组细胞；药物处理后进行96h释放的细胞中,MN-γ-H2AX(+)的发生频率几乎恢复到本底水平。 2)根据药物处理后,细胞核中γ-H2AX染色的情况将细胞分为三类：对于整个细胞核均呈现出均匀弥散性γ-H2AX染色的细胞,我们将其定义为一类细胞；对于细胞核中存在大量γ-H2AX焦点的细胞,我们将其定义为二类细胞；对于细胞核中几乎没有γ-H2AX信号的细胞,我们将其定义为三类细胞。结合各类细胞占总细胞的比例以及细胞周期结果推测,药物处理后所检测到的一类细胞与二类细胞很有可能是处于S期且经历DNA复制压力的细胞。药物处理后对不同释放时间各类细胞产生的微核数进行统计,结果发现,使用阻止DNA复制的药物处理后,未经释放的情况下,大多数新产生的MN-γ-H2AX(+)都是由处于S期的一类细胞与二类细胞产生的。由此明确了MN-γ-H2AX(+)是在S期形成的。 4.免疫荧光实验观察小鼠皮肤成纤维细胞(MSF)发现有γ-H2AX信号在细胞核外周聚集成簇并且有向外突出的趋势,推测这样的细胞核突出部位很可能是MN-γ-H2AX(+)的前体。 5.使用具有DNA复制遗传缺陷的细胞进行实验,结果发现： 1)通过流式细胞术和BrdU掺入实验证明,在RPA1低表达的MCF-7细胞中存在DNA复制压力。 2)通过微核计数发现,与对照细胞相比,RPA1(?)(?)表达细胞中MN-γ-H2AX(+)的发生频率有所增高。综上所述,MN-γ-H2AX(+)是一种新型微核,它不同于传统意义上有丝分裂后期形成的微核,主要是在细胞分裂间期形成的。我们推测这类微核的形成原因是细胞在S期遭遇DNA复制压力,进而导致了大量的DNA双链断裂损伤,这些无法修复的损伤部位聚集在一起排出细胞主核,形成MN-γ-H2AX(+)。研究这类新型的MN-γ-H2AX(+)有助于我们认识DNA复制压力所引起的基因组不稳定性,以及评估导致DNA复制压力的遗传和环境因素。第二部分活性氧(ROS)导致MN-γ-H2AX(+)发生频率增高第一部分的研究证明DNA复制压力能够诱发MN-γ-H2AX(+),且有多项研究证明ROS能够导致不同形式的DNA损伤,因此我们接下来研究了ROS水平与MN-γ-H2AX(+)形成之间的关系。我们使用药物影响ROS水平以及使用具有抗氧化基因缺陷的细胞进行实验,检测MN-γ-H2AX(+)的发生频率；同时使用具有抗氧化作用的药物对以上细胞进行联合处理,以抑制细胞内ROS水平的增高,分析ROS与MN-γ-H2AX(+)形成的关系。 1.使用导致ROS水平增高的药物过氧化氢(H202)以及具有抗氧化作用的药物N-乙酰半胱氨酸(NAC)处理细胞,结果如下： 1)使用不同浓度的H2O2(50μM,100μM,150μM)处理U20S细胞不同时间(2h,6h,12h,24h),随后更换为新鲜完全培养基释放48h,检测MN-γ-H2AX(+)的形成情况。结果显示,H202能够诱发MN-γ-H2AX(+),且呈剂量依赖性和时间依赖性。 2)使用具有抗氧化作用的药物NAC与H202联合处理细胞,结果发现,NAC能够下调H202所造成的MN-γ-H2AX(+)发生频率的增高。从而通过药物实验证明了ROS能够诱发MN-γ-H2AX(+)。 2.通过不同方式使具有抗氧化作用的基因p53功能改变后,检测MN-γ-H2AX(+)发生频率的变化情况。结果如下： 1)使用p53激活剂nutlin-3处理U20S细胞,MN-γ-H2AX(+)的发生频率有所降低。 2)使用p53抑制剂pifithrin-α处理U20S细胞,MN-γ-H2AX(+)的发生频率有明显增高。 3)使用p53功能缺陷的U20S细胞以及p53缺失的小鼠皮肤成纤维细胞进行实验,结果发现：与对照细胞相比,p53功能缺陷的细胞中,MN-γ-H2AX(+)的发生频率有明显增高。 4)使用具有抗氧化作用的药物NAC处理p53功能缺陷细胞及其对照细胞,结果发现：NAC能够下调由于p53功能缺陷而导致的MN-γ-H2AX(+)发生频率的增高。 3.为了了解p53下游基因对MN-γ-H2AX(+)形成的影响,我们使用siRNA抑制p53下游具有抗氧化作用的基因SESNl的表达。结果发现,与对照细胞相比,SESN1低表达的细胞中,MN-γ-H2AX(+)的发生频率有所增高。 4.有文献报道p400受到抑制后,细胞内ROS水平有所增高。因此,我们使用p400低表达的U20S细胞进行实验,结果发现p400表达量降低后,MN-γ-H2AX(+)的发生频率明显增高。进一步证明了ROS能够诱发MN-γ-H2AX(+)。综上所述,ROS水平增高能够诱发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 (+).

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：Q343;R394.3

【相似文献】