【作者】 金戈；

【导师】 董子明； 吴景兰；

【作者基本信息】 郑州大学 ， 病理与病理生理学， 2003， 博士

【摘要】 随着对肿瘤发病机制的研究，人们越来越多的认识到DNA修复酶类在维护细胞基因组稳定中作用的重要性，DNA修复酶类异常与肿瘤发生的关系日益受到人们的关注，目前，已经成为当前癌症病因学研究的一个热点。 碱基切除修复酶(base excision repair，BER)是主要的DNA修复酶类之一。它能够清除由各种内源性和外源性因素引起的基因组DNA 2-6个核苷酸损伤和无碱基位点(apurinic／apyrimidinic，AP)，维持基因组稳定性。DNA聚合酶β(polymerase β，pol β)主要是单个碱基切除修复中的关键酶之一。pol β广泛存在于真核和原核生物中。在发现的同类酶中分子量最小，在不同种属间具有高度保守性。在整个细胞周期中活性很低且不随细胞周期变动除去参与填充短片段(单个)核苷酸缺口，维持基因组稳定以外还可能参加DNA复制、重组以及癌细胞对某些药的抗药性，其突变和表达异常可能具有肿瘤启动作用。许多研究者在多种肿瘤组织和细胞系中发现了DNA pol β的基因突变，哺乳动物细胞DNA pol β的高表达可诱导染色体不稳定性增加，患癌风险增加。但是有关pol β突变和高表达与基因组稳定性的关系以及在癌变中的作用方面研究仍处于起步阶段，仍然需要进一步研究探讨。建立pol β稳定转染细胞系并将其应用于研究是一个有效的途径。至今建立的pol β稳定转染细胞系很少，并且国外同类研究主要集中在小鼠pol β基因，与人类pol β基因稳定转染细胞系相关的研究尚未见报道。 本实验将不同类型的DNA聚合酶β的的真核表达载体转染哺乳动物细胞，初步观察野生型和突变型DNA聚合酶β基因的表达对细胞生物学特性的影响，郑州大学2004年博士论文分化诱导剂和不同类型的人DNA聚合酶p基因转染对细胞的生物学效应为今后进一步研究由DNA聚合酶p突变引起的修复缺陷与肿瘤的发生发展的关系奠定基础。 肿瘤的发生是细胞的增殖分化失调的结果，而细胞增殖和分化的调控是多基因在多环节上控制的复杂过程，受到多种因素的影响。 诱导分化是指恶性肿瘤细胞在体内外分化诱导剂作用下，趋向正常细胞方向分化逆转的过程。癌细胞的诱导分化和分化治疗作为肿瘤治疗的一条新的途径，近年来研究非常活跃。 在诱导分化过程中许多与肿瘤发生关系密切的癌基因和抑癌基因的表达皆可发生变动，其中野生型p53(wild tyPe p53，叭p53)起关键作用。我们应用两种诱导分化剂作用于人食管癌Eca109细胞系，观察在细胞出现分化表型时DNA聚合酶p的表达和功能变动。 本研究分为以下两个部分:1，分化诱导剂全反式维甲酸(all.transretinoic acid，ATRA)、二甲亚矾(d加e御1 sulfoxide，DMSo)对Eca一109细胞的诱导分化作用及其与DNA聚合酶p之间的 关系;2.不同类型的聚合酶p的的真核表达载体稳定转染哺乳动物细胞系的建立、鉴 定及细胞生物学特性研究;实验方法: 1.建立全反式维甲酸(ATRA)和二甲亚矾(D Mso)诱导的Eca-109细胞系和PcDNA3 .1一lp一PM3及PcDNA3.1一帆polp稳定转染MH3T3的细胞系，同时建立各对照组细胞系。2.应用台盼兰拒染试验及MTT法记录细胞生长曲线。3.应用随机引物制备Biotin标记的Bio一帆p53的cDNA探针。应用体外转录法将机polp DNA单酶切为线性模板，加SP6RNA聚合酶、rNTPs及Bi。一11一dUTP进行体外转录制备反义RNA探针，以DNA斑点印迹检测标记探针的灵敏度，进行polp原位杂交。4.定位观察表达信号并相对定量:油镜下观察各组细胞的原位杂交信号和免疫反应性(POLB一IR、PCNA一TR)的定位及其强弱级别积分值。同时以不加标记探针和不加特异性抗体的标本分别作为原位杂交和免疫组化的阴性对照。5.抽提细胞总RNA，逆转录一聚合酶链式反应(Rr-PCR)法检测po伟mRNA的表达。6.软琼脂克隆形成试验测定诱导分化加药组及对照组的克隆形成率、郑州大学2004年博士论文分化诱导剂和不同类型的人DNA聚合酶日基因转染对细胞的生物学效应抑制率。7.应用DNA酸变性一甲基绿一派朗宁GS染色计数增殖细胞/分化细胞比率。8.流式细胞术检测分化组和转染组及对照组的细胞周期。9.以免疫印迹比较各组细胞的POLB蛋白表达水平。10.提取诱导分化各组及对照组的细胞核蛋白，检测BER功能。实验结果: 一、不同分化诱导剂对Eca-109细胞的诱导分化作用及其与polp表达及功能之间的关系1.绘制细胞生长曲线:ATRA组(5 pm。比)和DMSO(l .5%)组细胞与对照 组相比，生长明显减慢，第4天差异具有显著性(p<0.05)。2.矶p53基因的表达:应用5 p moUL八TRA和1.5%DMSO诱导Eca一109细胞趋 向分化时，诚p53基因表达上调，与未加药组相比差异具有显著性(p<0 .01)。3.DNA polp基因mRNA、蛋白的表达:ATRA和DMso可以在基因水平和蛋 白水平降低polp的表达。4.ATRA及DMSO对Eca-109细胞软琼脂集落形成能力的影响:ATRA及 DMSO均显著抑制Eca-109细胞在软琼脂中形成集落的能力，与未加药组相 比差异具有显著性(p<0 .01)。ATRA的抑制率为89%，DMso的抑制率为 92%。对照组集落不但比药物组集落大，且单个集落中细胞数目更多。5.增殖细胞比率计数:与对照组相比，八T更多还原

【Abstract】 With study on mechanism of oncogenesis, the important role of maintaining genome stability played by DNA repair enzymes has been considered more and more. The relationship between anomaly of the DNA repair enzymes and oncogenesis has been concerned, which has become a hot spot for research on oncogenesis.Base excision repair enzyme is one of DNA repair enzymes. It can remove 2-6 nucleotide damage and AP sites (apurinic/apyrimidine site) induced by exogenous and endogenous factors. DNA polymerase B (pol B ) is the key enzyme for single base excision repair. The pol B widely exists in both prokaryotes and eukaryotes, and is one with the smallest molecular weight among homologous enzymes, and processes high conservative nature among species. The activity of pol B is very low and independent with the cell cycle change. Besides, the DNA pol B can fill the nick of short patch (one nt) and maintain the genome stability, and it yet can involve DNA replication, recombination and drug resistance in cancer cells. The mutation and deregulation of pol B expression could initiate oncogenesis. Multiple mutations of pol B gene in various kinds of tumor tissues/cell lines were found, overexpression of pol B in mammalian cells could induce genome instability and incidental to oncogenesis. However, the correlation of overexpression and mutation of pol B with the genome stability and the its effect on oncogenesis has been explored primarily and needs further studies. The cell line established with stable transfection of pol B genesis available for study, the cell line establishment of stable transfection with pol B gene was rarely reported and the related research mainly focused on the mouse pol B abroad. The associated report with the cell line establishment of stable transfection with heterologous human pol B genes has not yet been reported.The constructed eukaryotic expression vectors carrying respective wild type and the mutant type DNA polymerase B were transfected into the mammalian cell and their biological effects were observed primarily. It will be a basis for further research on the relationship between the damage- repair from the DNA pol B mutation and the oncogenesis.The deregulation of differentiation and proliferation may result in carcinogenesis. The cell proliferation / differentiation is controlled by multiple genes regulated in multiple steps affected by various factors.Induction of differentiation refers to a reversible process of the malignant cancer cells induced through exogenous and endogenous differentiation reagents towards the normal. Differentiation-induction and its therapy for cancer disease, as a new strategy, had been studied actively in recent years.During the process of differentiation-induction, various kinds of oncogenes and antioncogenes may be altered correspondingly. By using all-trans retinoic acid( ATRA) and dimethyl sulfoxide( DMSO) as differentiation-inducing reagents the DNA polymerase B gene expression and its associated function were explored in human esophageal cancer 109 cells.This research contents can be divided into two parts:1. The effects on the human Eca-109 cell differentiation induced by all-trans retinoic acid(ATRA) and dimethyl sulfoxide(DMSO) and associated with the expression and function of DNA polymerase B .2. Establishment and identification of cell lines stably transfected with heterologous DNA polymerase B gene and the biological effects on NIH3T3 cells.Methods:1. The Eca-109 cell line treated with differentiation reagents and the NIH-3T3cell line stably transfected with pcDNA3.1-pol B -PM3 and pcDNA3.1-wtpol B and their corresponding control cell lines were established respectively. 2. The growth curve of the pol B gene transfected into NIH3T3 cells was drawn by the MTT assay or trypan blue exclusion assay. 3. The Biotin-labeled wtp53 cDNA probe was prepared by random primer method. The Bio-labeled wtpol B antisense RNA probe was prepared by transcription in vitro. The transcription in vitro was carried out by addition of SP6 RNA polymera更多还原