甲基丙烯酸环氧丙酯致人支气管上皮细胞恶性转化相关DNA甲基化的研究

发布时间：2018-07-01 09:14

本文选题：甲基丙烯酸环氧丙酯 + 人支气管上皮细胞　；参考：《中国疾病预防控制中心》2012年硕士论文

【摘要】：DNA甲基化是指由DNA甲基转移酶(DNMT)催化,将活性甲基从S-腺苷甲硫氨酸(SAM)转移至胞嘧啶的5位碳原子上,形成5-甲基胞嘧啶的化学修饰过程,是调节真核生物基因表达的重要方式之一。与经典遗传理论不同的是,DNA甲基化是不改变DNA序列的遗传现象,并可受外界多种因素的干扰发生改变。研究发现,DNA甲基化与人类多种肿瘤的发生发展密切相关,广泛参与细胞恶性转化过程及基因调控、细胞增殖、分化、发育、等生物学过程,具有调控相关癌基因、抑癌基因等多种基因表达的功能,被认为是致癌物既往暴露和癌症发病风险中新兴的最有前景的肿瘤分子生物标志物(biomarker)。肿瘤细胞中正常DNA甲基化模式的异常改变,表现为全基因组广泛低甲基化及抑癌基因、癌基因、凋亡相关基因、错配修复基因等多类基因的高甲基化,这种基因异常甲基化状态的改变对维持正常细胞功能、遗传印记以及人类肿瘤的发生发展产生重要影响,对不同肿瘤相关基因甲基化状态改变的研究可为肿瘤发生机制研究及其治疗提供新途径。甲基丙烯酸环氧丙酯(Glycidyl Methacrylate,简称GMA),是丙烯酸酯胶的主要组分,含有碳碳双键和环氧基团,可参与离子型和自由基型反应,具有很高的反应活性,其合成产品兼具丙烯酸的耐候性和环氧的耐化学性。GMA作为一种聚合用单体,具有良好的防紫外、耐水和耐热等特性,在树脂、涂料、粘合剂、塑料等工业中均得到广泛应用。以往研究表明,GMA属低毒类、具有明显的刺激性、引起速发型和迟发型变态反应、体外多项致突变试验结果呈阳性、具生殖发育毒性,并可诱导BALB/C3T3细胞、金仓鼠细胞(SHE)、正常人胚肺成纤维(KMB-13)细胞等多种哺乳类细胞发生恶性转化,具有潜在致癌性。细胞体外转化试验是研究各种理化因素致癌机制的重要方法,由于人类80%肿瘤源于上皮组织,直接以人的上皮细胞为背景进行肿瘤发生发展的试验研究,在种属和组织差异方面更具优势。16HBE细胞(human bronchial epithelial cells)是经SV40Large T抗原永生化的人支气管上皮细胞系,具有正常人呼吸道上皮细胞的形态和功能,该细胞体外易于培养,且裸鼠体内无致瘤性,以其作为转化试验的靶细胞可在体外模拟体内细胞受致癌原作用后向肿瘤细胞演变的过程。本研究以8μg/ml GMA染毒16HBE细胞(DMSO作为溶剂对照),每次染毒72h,间隔24h,连续染毒3次,染毒结束后计为第1代转化细胞,连续培养细胞至第30代,期间采用刀豆凝集素A(conA)凝集试验和锚着非依赖性(AIG)试验对细胞恶性转化生物学特性进行鉴定。收集第10代(转化前期)、第20代(转化中期)、第30代(转化后期)转化细胞,采用NimbleGen公司生产的"NimbleGen HG18CpG Promoter Microarray Methlation"甲基化芯片动态分析GMA致16HBE细胞恶性转化过程中不同时期相关甲基化基因,并对部分基因甲基化状态进行验证,探讨GMA致16HBE细胞恶性转化的DNA甲基化机制。 1.GMA致人支气管上皮细胞恶性转化及生物学特性鉴定 GMA组第14代转化细胞开始对conA的凝集敏感性增强；第20代转化细胞表现出凝集速度加快,凝集度增高现象,随着conA浓度的增加和时间的延长,细胞凝集速度加快,镜下可见细胞凝集成团,且在低浓度conA剂量下也出现凝集,同代龄DMSO组细胞分散均匀,30min尚未出现凝集现象。第10代、14代及20代转化细胞在软琼脂中均未形成集落,第30代转化细胞可在软琼脂中形成细胞集落,而同代龄DMSO组细胞未形成集落,提示第30代转化细胞已具备恶性转化细胞生物学特性。 2.GMA致16HBE细胞恶性转化过程中甲基化基因分析 2.1GMA致16HBE细胞恶性转化过程中各时点甲基化基因分析 GMA组有1374个基因在转化前期甲基化；825个基因在转化中期甲基化；1149个基因在转化后期甲基化；30个基因在转化前期、中期及后期均甲基化。 2.2GMA致16HBE细胞恶性转化过程中不同时点甲基化基因状态变化分析 GMA组有318个基因转化前期甲基化而转化中、后期未见甲基化；272个基因转化中期甲基化而转化前、后期未见甲基化；683个基因转化后期甲基化而转化前、中期未见甲基化；73个基因转化前期、中期甲基化而转化后期未见甲基化；67个基因转化前期、后期甲基化而转化中期未见甲基化；59个基因转化中期、后期甲基化而转化前期未见甲基化。 3.GMA致16HBE细胞恶性转化过程中相关基因甲基化状态分析结合上述甲基化芯片结果及相关文献对P15、OPCML、THBS1基因在不同时期甲基化状态进行分析。结果显示,OPCML基因在GMA诱导细胞转化不同阶段均甲基化；THBS1基因在GMA诱导细胞转化前期甲基化而转化中期、后期未见甲基化；P15基因在GMA诱导细胞转化中期、后期甲基化而转化前期未见甲基化,与甲基化芯片分析结果一致。结果提示,OPCML基因可能是GMA诱导细胞恶性转化过程的相关甲基化基因,THBSl基因可作为GMA诱导细胞恶性转化的早期敏感指标,而P15基因甲基化状态与细胞恶性程度相关,可将其作为细胞恶性转化的特异性基因。综上所述,本研究探讨GMA致16HBE细胞恶性转化过程中转化前期、中期和后期相关基因甲基化状态的改变,并对部分基因甲基化状态进行检测。结果提示,GMA致16HBE细胞恶性转化过程中不同阶段基因DNA甲基化状态发生改变,DNA甲基化这种基因修饰作用在细胞恶性转化过程中伴随细胞转化发生变化,与相关基因的表达及其功能改变密切相关。OPCML基因可能是GMA诱导细胞恶性转化过程的相关甲基化基因,THBS1基因可作为GMA诱导细胞恶性转化的早期敏感指标,而P15基因甲基化状态与细胞恶性程度相关,可将其作为细胞恶性转化的特异性基因。本研究工作为进一步探讨GMA致16HBE细胞恶性转化及相关分子标志物的筛选奠定了基础。
[Abstract]:DNA methylation is a DNA methyl transferase (DNMT) catalyzed by the transfer of active methyl from S- adenosine methionine (SAM) to 5 carbon atoms of cytosine, forming a chemical modification process of 5- methyl cytosine, which is one of the important ways to regulate eukaryotic gene expression. Unlike the classic genetic theory, DNA methylation does not change the order of DNA. It is found that DNA methylation is closely related to the development of a variety of human tumors, and is widely involved in the process of cell malignant transformation and gene regulation, cell proliferation, differentiation, development, and other biological processes, and has a variety of gene tables that regulate the related oncogenes and tumor suppressor genes. The function of it is considered to be the most promising biomarker (biomarker) of the carcinogens in the past and cancer risk. Abnormal changes in normal DNA methylation patterns in tumor cells are characterized by extensive genomics, extensive hypomethylation and tumor suppressor genes, oncogenes, apoptosis related genes, mismatch repair genes, and so on. The hypermethylation of genes, the alteration of the methylation status of this gene, has an important effect on maintaining normal cell function, genetic imprinting and the development of human tumor. The study on the change of methylation status of different tumor related genes can provide a new way for the study and treatment of tumor pathogenesis.
Glycidyl Methacrylate (GMA) is the main component of acrylate adhesive. It contains carbon carbon double bond and epoxy group. It can participate in ionic and radical reaction. It has high reactive activity. The synthetic product has the weatherability of acrylic acid and the chemical resistance of epoxy.GMA as a monomer for polymerization. It has good anti UV, water resistance and heat resistance and other properties. It has been widely used in resins, coatings, adhesives, plastics and other industries. Previous studies have shown that GMA is a low toxic type, with obvious irritation, rapid and delayed allergy. The results of multiple mutagenesis in vitro are positive, with reproductive toxicity and can induce BALB/C3T. 3 cells, golden hamster cells (SHE), normal human embryonic lung fibroblast (KMB-13) cells and other mammalian cells undergo malignant transformation, which has potential carcinogenicity.
In vitro transformation test of cells is an important method to study the carcinogenic mechanism of various physical and chemical factors. Because human 80% tumors are derived from epithelial tissue, human epithelial cells are used directly as the background to study the development of tumor. The more dominant.16HBE cells (human bronchial epithelial cells) in the species and tissue differences are SV40Large The T antigen immortalized human bronchial epithelial cell line has the morphology and function of normal human respiratory epithelial cells. This cell is easily cultured in vitro, and there is no tumorigenicity in the nude mice. As a target cell of the transformation test, it can simulate the process of cell evolution to the tumor cells after the oncogenic action of the cells in vitro.
In this study, 16HBE cells infected with 8 g/ml GMA (DMSO as a solvent control) were poisoned each time with 72h, interval 24h, and continuously poisoned for 3 times. After the end of the poisoning, the first generation of transformed cells were counted, and the cells were continuously cultured to thirtieth generations. During the period, the biological characteristics of cell malignant transformation were carried out by the agglutination test of lectin A (conA) and the anchoring non dependent (AIG) test. Identification. The tenth generation (early transformation), the twentieth generation (metaphase), the thirtieth generation (late transformation) were transformed into cells. The NimbleGen HG18CpG Promoter Microarray Methlation methylation chip produced by the company was used to dynamically analyze the methylation genes in the malignant transformation of 16HBE cells from GMA, and some of the methylated genes were methylated by the GMA Promoter Microarray Methlation methylation chip. To verify the DNA methylation mechanism of GMA induced malignant transformation of 16HBE cells.
Malignant transformation and biological characteristics of human bronchial epithelial cells induced by 1.GMA
The fourteenth generation of transformed cells in group GMA began to increase the agglutination sensitivity of conA, and the twentieth generation of transformed cells showed agglutination faster and more agglutination. With the increase of conA concentration and time, the cell agglutination speed was accelerated. The cell agglutination was observed under the microscope, and the agglutination of the cells was also agglutinated under the low concentration of conA, and the DMSO group of the same age group was fine. In the tenth generation, the 14 generation and the 20 generation of the transformed cells in the soft agar, the tenth generation, the thirtieth generation cells can form the cell colony in the soft agar, but the cells of the same generation DMSO group have not formed a colony, suggesting that the thirtieth generation of transformed cells have been prepared for the biological characteristics of malignant transformation cells.
Analysis of methylation genes in malignant transformation of 16HBE cells induced by 2.GMA
Analysis of methylation genes at different time points in malignant transformation of 16HBE cells induced by 2.1GMA
In group GMA, 1374 genes were methylated in the early stage of transformation; 825 genes were methylated in the metaphase; the 1149 genes were methylation at the later stage of transformation; the 30 genes were methylation at the early stage, middle and later stage.
Analysis of methylation status at different time points in malignant transformation of 16HBE cells induced by 2.2GMA
In group GMA, 318 genes were transformed by methylation in the early stage, and no methylation was found at the later stage. The 272 genes were converted into methylation in the middle period and no methylation was found in the later stage. The 683 genes were converted to methylation in the late stage and no methylation in the middle period. The 73 genes were transformed earlier, middle stage methylation and later transformation were not methylation; 67 bases were found. In the early stage of transformation, there was no methylation in the metaphase of metaphase transformation. In the middle stage of the 59 gene transformation, there was no methylation in the late stage of transformation.
Analysis of methylation status of related genes in malignant transformation of 16HBE cells induced by 3.GMA
The methylation status of P15, OPCML, THBS1 gene was analyzed at different stages with the results of the methylation chip and related literature. The results showed that the OPCML gene was methylation at different stages of GMA induction of cell transformation, and the THBS1 gene was methylation at the early stage of induction of cell transformation by GMA and no methylation at the later stage, and P15 gene was induced in GMA to induce GMA. It is suggested that the OPCML gene may be a related methylation gene in the process of cell malignant transformation induced by GMA, and that the THBSl gene can be used as an early sensitive index for GMA induced malignant transformation of cells, and the methylation status of the P15 gene and the finer gene of the P15 gene. The degree of cell malignancy is related, and can be used as a specific gene for malignant transformation of cells.
To sum up, this study investigates the changes in the methylation status of the related genes in the prophase, medium-term and late transformation of GMA induced malignant transformation of 16HBE cells and to detect the methylation status of some genes. The results suggest that the DNA methylation status of different stages in the malignant transformation of 16HBE cells induced by GMA and the base of DNA methylation The change of cell transformation in the process of cell malignant transformation is associated with the expression of related genes and their function changes. The.OPCML gene may be a related methylation gene in the process of cell malignant transformation induced by GMA. The THBS1 gene can be used as an early sensitive indicator of GMA induced cell malignant transformation, and the methylation of the P15 gene. The state is related to the degree of cell malignancy, which can be used as a specific gene for malignant transformation of cells. This work has laid a foundation for further exploration of the malignant transformation of 16HBE cells induced by GMA and the screening of related molecular markers.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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