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题名: 小分子化合物调控DNA 氧化及胚胎干细胞低甲基化研究
作者: 李翠平
学位类别: 博士
答辩日期: 2016-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 汪海林
关键词: DNA碱基损伤,毛细管电泳-激光诱导荧光(CE-LIF),维生素C,PD0325901,低甲基化 ; Oxidized DNA bases, Capillary electrophoresis laser-induced fluorescence (CE-LIF), Vitamin C, PD025901, Hypomet
其他题名: DNA oxidative damage analysis and DNA hypomethylation regulation of embryonic stem cells by small-molecule compounds
学位专业: 环境科学
中文摘要:     本论文分为两部分内容:1. DNA修复酶结合毛细管电泳-激光诱导荧光高灵敏检测DNA氧化碱基损伤;2. MEK抑制剂PD0325901联合维生素C协同诱导小鼠胚胎干细胞低甲基化研究。
自由基的形成既与内源的细胞正常有氧代谢和炎症过程有关,还与一些外源因素相关,比如紫外照射、离子辐射、氧化还原的药物等。自由基能够与DNA分子反应,形成一系列的氧化损伤产物。而癌症的发生以及衰老及相关的疾病,比如阿尔茨海默症、帕金森症和肌萎缩侧索硬化症,被认为与DNA氧化损伤密切相关。因此,快速、准确、灵敏的DNA氧化损伤方法的建立对于理解和临床诊断氧化损伤相关疾病具有重要的指导意义。
    毛细管电泳-激光诱导荧光(CE-LIF)具有高效快速分离、样品消耗量小、灵敏度高的优点,在DNA氧化损伤的分析中已有应用;DNA修复酶能够识别并切除特定的DNA氧化损伤,广泛应用于利用DNA断裂检测DNA氧化损伤的方法中;量子点(QD)具有荧光量子产率高,抗光漂白等独特的光学性质,是一种很有应用前景的高灵敏分析的光学标记物。
    本文第2章将选择性的DNA修复酶与高荧光强度的量子点标记相结合,发展了一种新的高灵敏的利用毛细管电泳-激光诱导荧光测定氧化碱基损伤的方法。氧化的DNA碱基首先被DNA碱基切除修复糖基化酶识别并且切除,在氧化位置留下一个脱嘌呤或脱嘧啶位点(AP sites),进一步利用糖基化酶的AP-裂解酶活性将AP sites两端的磷酸二酯键切断,产生一个5'-和3'-磷酸基团的核苷酸空位。碱性磷酸酶将核苷酸空位两端的5'-和3'磷酸去掉,而后DNA聚合酶在核苷酸空位处引入生物素(biotin)修饰的ddNTP,使DNA碱基损伤转换为biotin标记的位点。Biotin标记的DNA进一步与链酶亲合素(streptavidin)标记的量子点通过biotin-streptavidin之间的非共价作用结合,然后利用CE-LIF分析。结合了DNA的量子点和过量的自由的量子点通过毛细管电泳能够得到有效分离,结合在线激光诱导荧光能够实现高灵敏分析。利用合成的biotin标记的DNA标准品,该方法的检出限(S/N=3)从量的角度计算为1.1×10-19 mol,从浓度的角度计算为2.9 pM,灵敏度远高于常用的氧化损伤检测方法:高效液相色谱-电化学检测方法(HPLC-ECD)。该方法能够用于Fenton反应和紫外照射中痕量的DNA氧化损伤的检测。甲酰胺嘧啶[fapy]-DNA糖基化酶(FPG)主要识别的底物是8-羟基-鸟嘌呤,它被认为是DNA氧化损伤的标志物,与CE-LIF结合能够测定其含量。另外,CE-LIF与切除嘧啶损伤的内切酶VIII DNA糖基化酶结合可以定量嘧啶损伤的量。因此,该方法是一种快速、灵敏、能够用于多种氧化损伤分析的方法。
    胚胎干细胞(embryonic stem cells,ES cells)是源于囊胚的内层细胞团(the inner cell mass,ICM),具有多能性和自我更新的能力。多能性指的是细胞分化成所有体细胞和生殖细胞的能力。ES细胞的建立为体外研究胚胎干细胞的发育过程提供了机会,并且因为其具有多能性,诱导分化成的体细胞能够服务于再生医学,因此胚胎干细胞的体外培养具有十分重大的意义。
    传统的血清培养方式下,胚胎干细胞展现出多样性,分成类似于ICM和倾向于分化的两个群体,并且其基因组DNA呈现整体高甲基化。但是,ICM基因组DNA呈现整体低甲基化,并且其多能性与DNA的低甲基化状态密切相关。最近有大量小分子化合物引导细胞命运的报道,表明可以利用化学的方法控制细胞,实现特定的目标。
    本文第3章,我们首次提出了维生素C联合MEK抑制剂PD0325901快速、有效实现小鼠胚胎干细胞低甲基化的新方法。两个小分子化合物的结合能够在5天内促使小鼠胚胎干细胞中5-甲基胞嘧啶(5-methylcytosine,5mC)的含量下降90%,即从3.2 5mC/100 C下降到~0.3 5mC/100 C。降低90%后的甲基化水平与原生殖细胞(primordial germ cells,PGCs)的甲基化水平相当。PGC的多能性与DNA全面低甲基化密切相关。相比之下,维生素C或者PD0325901单独处理小鼠ES细胞仅造成一定幅度的DNA甲基化损失。我们的机理研究表明PD0325901促进Prdm14的蛋白表达,Prdm14抑制从头合成的DNA甲基转移酶Dnmt3b以及它的辅助因子Dnmt3l的蛋白表达,从而从源头上抑制5mC的合成。维生素C的加入促使Tet1/Tet2 (ten-eleven translocation)双加氧酶催化氧化的5mC氧化产物显著增加,这一点从5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5hmC)水平的显著提升可以看出。但是,一旦敲除Tet1/Tet2,维生素C失去了增强PD0325901刺激的小鼠ES细胞基因组DNA低甲基化的功能。小鼠胚胎干细胞在补充了维生素C和PD0325901两种小分子化合物的培养基中展现出更好的形态和多能性。因此,我们论证了一种通过促进DNA去甲基化和抑制DNA甲基化的合成,协同促进小鼠ES细胞低甲基化的新方法。
英文摘要:     The dissertation includes two parts: 1. Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis for oxidized bases. 2. MEK inhibitor PD0325901 and vitamin C synergistically induce hypomethylation of mouse embryonic stem cells.
    The formation of free radicals results from numerous of endogenous cellular metabolism in aerobic organisms and inflammatory reactions as well as from exogenous sources such as ionizing radiations, UV radiation and redox-cycling drugs. Free radicals can react with DNA molecules, resulting in a wide spectrum of damage products. Oxidatively generated DNA damage is considered to be one significant contributing factor to cancer, aging and age-related human diseases, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Therefore, a method with fast, accuracy and sensitivity was needed for the understanding and clinical diagnosis of the oxidative damage-caused diseases.
    Capillary electrophoresis, due to its features of highly efficient and rapid separation, high sensitivity and low sample consumption, has been applied in the analysis of oxidative DNA damage. DNA repair enzymes can recognize and excise oxidized bases and convert them into DNA strand breaks which in turn can be detected in a very sensitive manner. Quantum dots (QD) have unique optical properties such as high fluorescence quantum yields and photostabilities against bleaching and are the promising optical labels for sensitive analysis.
In Chapter 2, by taking advantage of selective enzymatic cleavage and quantum dot labeling, we developed a novel capillary electrophoresis-laser induced fluorescence method for sensitive detection of oxidized DNA bases. Oxidized DNA bases are firstly recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5'- and 3'- phosphate groups. After dephosphorylation of 5' and 3' with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within DNA strand by the DNA polymerase I. The biotin-tagged DNA is further labeled with QD-streptavidin conjugate through the non-covalent interaction. The DNA-bound QD is well separated from excess. DNA-unbound QD by highly efficient capillary electrophoresis and sensitively detected by the online coupled laser-induced fluorescence analysis. By the use of synthesized standard DNA, we showed that the approach has the low limits of detection of 1.1×10-19 mol in mass and 2.9 pM in concentration. In comparison with high performance liquid chromatography-electrochemical detection (HPLC-ECD), one of the common methods, our method exhibits higher sensitivity. By this method, we can assess the trace levels of oxidized DNA bases induced by Fenton reaction and UV irradiation. Interestingly, 8-OH-guanine which was commonly considered as a biomarker of oxidative DNA damage, was the major substrate of formamidopyrimidine glycosylase (FPG) protein and can be measured by CE-LIF, while endonuclease VIII allow for the detection of oxidized pyrimidine bases by CE-LIF. Therefore, an approach with fast, sensitivity and analysis of multiple DNA damage was established.
    Embryonic stem cells (ES cells) are pluripotent and selfrenewing cells derived from the inner cell mass (ICM) of blastocysts. Pluripotency refers to the capacity of forming all the somatic lineages as well as the germline. Establishment of ESCs can provide the chance to investigate the developmental processes in vitro. Furthermore, due to their pluripotency, various cell types can be generated for regenerative medicine. Therefore, ES cells cultured in vitro are of great significance.
    Traditionally, ES cells are maintained in serum, however, under the conditions, ES cells show heterogeneity. ES cells consist of two populations, one similar to the naive ICM state and the other similar to the primed epiblast state. Furthermore, serum-cultured ES cells often exhibit global genomic DNA hypermethylation. Interestingly, global erasure of genomic 5mC occurs in ICM, and the hypomethylation in ICM is closely linked with pluripotent state. Recently, many studies have shown that a combination of small-molecule chemicals can guide cell fates, suggesting an open chemistry way to manipulate cells to achieve a specific goal.
    In Chapter 3, we for the first time demonstrate that a combination of vitamin C (Vc) and MEK inhibitor PD0325901 can rapidly and effectively achieve hypomethylation in mouse embryonic stem cells (ES cells). The combination of two small-molecule compounds can promote about 90% erasure of 5-methylcytosine (5mC) within 5 days (decreasing from 3.2 to ~0.3 5mC per 100 C) in mouse embryonic stem cells. The hypomethylated level is comparable to that of gonadal primordial germ cells (PGCs), whose pluripotency is closely associated with the global DNA hypomethylation. In contrast, Vc or PD0325901 alone only induces a moderately reduced level of global DNA methylation. Our mechanistic study suggested that PD0325901 elevated expression of Pdrm14, which repressed de novo methyltransferase Dnmt3b and its cofactor Dnmt3l at levels of protein, via the mode to eliminate 5mC from de novo synthesis. By further addition of Vc, the oxidation of 5mC as catalyzed by Tet1/Tet2 dioxygenases was significantly increased as manifested by the elevated level of 5-hydroxymethylcytosine. However, by the depletion of Tet1/Tet2, Vc failed to enhance PD0325901-stimulated hypomethylation of ESCs’ genomic DNA. Furthermore, mouse ESCs in Vc/PD0325901-supplemented medium show great morphology and pluripotency. Therefore, we demonstrated a synergistic mechanism of enhancing DNA demethylation and repressing DNA methylation simultaneously in ES cells and developed a novel approach for maintaining ES cells at a hypomethylated state.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/36873
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
李翠平. 小分子化合物调控DNA 氧化及胚胎干细胞低甲基化研究[D]. 北京. 中国科学院研究生院. 2016.
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