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  本文選題：甲基丙烯酸環(huán)氧丙酯 + 人支氣管上皮細胞　； 參考：《中國疾病預防控制中心》2012年碩士論文

【摘要】：DNA甲基化是指由DNA甲基轉移酶(DNMT)催化,將活性甲基從S-腺苷甲硫氨酸(SAM)轉移至胞嘧啶的5位碳原子上,形成5-甲基胞嘧啶的化學修飾過程,是調(diào)節(jié)真核生物基因表達的重要方式之一。與經(jīng)典遺傳理論不同的是,DNA甲基化是不改變DNA序列的遺傳現(xiàn)象,并可受外界多種因素的干擾發(fā)生改變。研究發(fā)現(xiàn),DNA甲基化與人類多種腫瘤的發(fā)生發(fā)展密切相關,廣泛參與細胞惡性轉化過程及基因調(diào)控、細胞增殖、分化、發(fā)育、等生物學過程,具有調(diào)控相關癌基因、抑癌基因等多種基因表達的功能,被認為是致癌物既往暴露和癌癥發(fā)病風險中新興的最有前景的腫瘤分子生物標志物(biomarker)。腫瘤細胞中正常DNA甲基化模式的異常改變,表現(xiàn)為全基因組廣泛低甲基化及抑癌基因、癌基因、凋亡相關基因、錯配修復基因等多類基因的高甲基化,這種基因異常甲基化狀態(tài)的改變對維持正常細胞功能、遺傳印記以及人類腫瘤的發(fā)生發(fā)展產(chǎn)生重要影響,對不同腫瘤相關基因甲基化狀態(tài)改變的研究可為腫瘤發(fā)生機制研究及其治療提供新途徑。 甲基丙烯酸環(huán)氧丙酯(Glycidyl Methacrylate,簡稱GMA),是丙烯酸酯膠的主要組分,含有碳碳雙鍵和環(huán)氧基團,可參與離子型和自由基型反應,具有很高的反應活性,其合成產(chǎn)品兼具丙烯酸的耐候性和環(huán)氧的耐化學性。GMA作為一種聚合用單體,具有良好的防紫外、耐水和耐熱等特性,在樹脂、涂料、粘合劑、塑料等工業(yè)中均得到廣泛應用。以往研究表明,GMA屬低毒類、具有明顯的刺激性、引起速發(fā)型和遲發(fā)型變態(tài)反應、體外多項致突變試驗結果呈陽性、具生殖發(fā)育毒性,并可誘導BALB/C3T3細胞、金倉鼠細胞(SHE)、正常人胚肺成纖維(KMB-13)細胞等多種哺乳類細胞發(fā)生惡性轉化,具有潛在致癌性。 細胞體外轉化試驗是研究各種理化因素致癌機制的重要方法,由于人類80%腫瘤源于上皮組織,直接以人的上皮細胞為背景進行腫瘤發(fā)生發(fā)展的試驗研究,在種屬和組織差異方面更具優(yōu)勢。16HBE細胞(human bronchial epithelial cells)是經(jīng)SV40Large T抗原永生化的人支氣管上皮細胞系,具有正常人呼吸道上皮細胞的形態(tài)和功能,該細胞體外易于培養(yǎng),且裸鼠體內(nèi)無致瘤性,以其作為轉化試驗的靶細胞可在體外模擬體內(nèi)細胞受致癌原作用后向腫瘤細胞演變的過程。 本研究以8μg/ml GMA染毒16HBE細胞(DMSO作為溶劑對照),每次染毒72h,間隔24h,連續(xù)染毒3次,染毒結束后計為第1代轉化細胞,連續(xù)培養(yǎng)細胞至第30代,期間采用刀豆凝集素A(conA)凝集試驗和錨著非依賴性(AIG)試驗對細胞惡性轉化生物學特性進行鑒定。收集第10代(轉化前期)、第20代(轉化中期)、第30代(轉化后期)轉化細胞,采用NimbleGen公司生產(chǎn)的"NimbleGen HG18CpG Promoter Microarray Methlation"甲基化芯片動態(tài)分析GMA致16HBE細胞惡性轉化過程中不同時期相關甲基化基因,并對部分基因甲基化狀態(tài)進行驗證,探討GMA致16HBE細胞惡性轉化的DNA甲基化機制。 1.GMA致人支氣管上皮細胞惡性轉化及生物學特性鑒定 GMA組第14代轉化細胞開始對conA的凝集敏感性增強；第20代轉化細胞表現(xiàn)出凝集速度加快,凝集度增高現(xiàn)象,隨著conA濃度的增加和時間的延長,細胞凝集速度加快,鏡下可見細胞凝集成團,且在低濃度conA劑量下也出現(xiàn)凝集,同代齡DMSO組細胞分散均勻,30min尚未出現(xiàn)凝集現(xiàn)象。第10代、14代及20代轉化細胞在軟瓊脂中均未形成集落,第30代轉化細胞可在軟瓊脂中形成細胞集落,而同代齡DMSO組細胞未形成集落,提示第30代轉化細胞已具備惡性轉化細胞生物學特性。 2.GMA致16HBE細胞惡性轉化過程中甲基化基因分析 2.1GMA致16HBE細胞惡性轉化過程中各時點甲基化基因分析 GMA組有1374個基因在轉化前期甲基化；825個基因在轉化中期甲基化；1149個基因在轉化后期甲基化；30個基因在轉化前期、中期及后期均甲基化。 2.2GMA致16HBE細胞惡性轉化過程中不同時點甲基化基因狀態(tài)變化分析 GMA組有318個基因轉化前期甲基化而轉化中、后期未見甲基化；272個基因轉化中期甲基化而轉化前、后期未見甲基化；683個基因轉化后期甲基化而轉化前、中期未見甲基化；73個基因轉化前期、中期甲基化而轉化后期未見甲基化；67個基因轉化前期、后期甲基化而轉化中期未見甲基化；59個基因轉化中期、后期甲基化而轉化前期未見甲基化。 3.GMA致16HBE細胞惡性轉化過程中相關基因甲基化狀態(tài)分析 結合上述甲基化芯片結果及相關文獻對P15、OPCML、THBS1基因在不同時期甲基化狀態(tài)進行分析。結果顯示,OPCML基因在GMA誘導細胞轉化不同階段均甲基化；THBS1基因在GMA誘導細胞轉化前期甲基化而轉化中期、后期未見甲基化；P15基因在GMA誘導細胞轉化中期、后期甲基化而轉化前期未見甲基化,與甲基化芯片分析結果一致。結果提示,OPCML基因可能是GMA誘導細胞惡性轉化過程的相關甲基化基因,THBSl基因可作為GMA誘導細胞惡性轉化的早期敏感指標,而P15基因甲基化狀態(tài)與細胞惡性程度相關,可將其作為細胞惡性轉化的特異性基因。 綜上所述,本研究探討GMA致16HBE細胞惡性轉化過程中轉化前期、中期和后期相關基因甲基化狀態(tài)的改變,并對部分基因甲基化狀態(tài)進行檢測。結果提示,GMA致16HBE細胞惡性轉化過程中不同階段基因DNA甲基化狀態(tài)發(fā)生改變,DNA甲基化這種基因修飾作用在細胞惡性轉化過程中伴隨細胞轉化發(fā)生變化,與相關基因的表達及其功能改變密切相關。OPCML基因可能是GMA誘導細胞惡性轉化過程的相關甲基化基因,THBS1基因可作為GMA誘導細胞惡性轉化的早期敏感指標,而P15基因甲基化狀態(tài)與細胞惡性程度相關,可將其作為細胞惡性轉化的特異性基因。本研究工作為進一步探討GMA致16HBE細胞惡性轉化及相關分子標志物的篩選奠定了基礎。
[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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R114

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