乳腺癌MDA-MB-231细胞不同培养模型的构建及DNA甲基化分析

发布时间：2018-04-02 12:20

本文选题：肿瘤　切入点：DNA甲基化　出处：《吉林大学》2016年硕士论文

【摘要】：背景与目的:乳腺癌是中国女性发病率最高的肿瘤。每年中国乳腺癌新发数量占全世界的12.2%;死亡数量占9.6%,是继肺癌、胃癌、肝癌、食管癌、大肠癌之后,第六大中国女性癌症死亡原因。肿瘤发生是体内多因素诱导并在体内的复杂环境逐步演化而成。认为基因突变的积累是肿瘤发生的主要原因,但随着表观遗传学的提出和越来越多的研究显示遗传学和表观遗传学的改变均可引起基因的异常表达。表观遗传学的改变对肿瘤的发生和进展十分重要。研究显示不同的细胞培养方法下肿瘤细胞的生物学行为表现也不同,且细胞能通过升高或降低基因启动子区域的甲基化影响基因功能从而控制细胞的性状和恶性度。但DNA甲基化状态的改变是否受细胞培养方法的影响尚不清楚。本研究通过构建2D、3D细胞培养和肿瘤原位移植模型,观察三种培养模型下肿瘤细胞的生长方式差别,探索不同模型所营造的生长环境对MDAMB-231细胞基因甲基化状态及相关细胞通路差异性。通过本研究结果进一步论述基因甲基化状态对肿瘤发生进展过程的影响。方法:构建二维(two dimensional,2D)即传统平面培养、三维(three dimensional,3D)细胞培养和小鼠原位移植模型(mouse orthotopic transplantation model,简称Tissue,Ti),采用三种模型培养MDA-MB-231细胞,观察三种模型下肿瘤细胞的生长方式,并收集细胞,使用基因组抽提试剂盒提取DNA,应用DNA甲基化450K芯片和genomestudio分析软件计算分析乳腺癌MDA-MB-231细胞基因组每个基因每个甲基化位点,分别用β值、Diff Score值、Delta_Beta表示。比较两种培养方法间乳腺癌MDA-MB-231细胞基因组甲基化状态筛选差异的甲基化基因,并运用DAVID在线分析工具对其进行基因本体论分析和细胞通路分析。结果:1、二维细胞培养的乳腺癌MDA-MB-231细胞贴壁单层生长,细胞呈梭形,仅存在细胞与细胞之间的缝隙连接。三维细胞培养的乳腺癌MDA-MB-231细胞,呈星球状生长,由单个细胞形成克隆团块,存在细胞与细胞间及细胞与细胞外基质间的连接。小鼠原位移植的MDA-MB-231细胞在小鼠皮下脂肪垫部位生长出一个肿瘤团块,性状类似真实的肿瘤组织。2、分别经过二维和三维细胞培养后的MDA-MB-231肿瘤细胞查出480个甲基化状态有差异的基因(P0.05)。三维培养和肿瘤原位移植模型间的MDA-MB-231细胞则发现86448个有差异基因(P0.05),而二维细胞培养与肿瘤原位移植模型间的MDA-MB-231细胞有90005个差异基因(P0.05)。3、三维细胞培养与二维细胞培养、三维细胞培养与肿瘤原位移植模型、肿瘤原位移植模型与二维细胞培养间的差异性甲基化基因在基因本体论部分条目上均有相同富集,分别是多细胞生物体发育(P0.05)和细胞分化(P0.05)。三种模型间差异甲基化基因在细胞通路上的富集也存在部分重合,如MAPK信号通路(P0.05)、细胞黏附分子作用通路(P0.05)、肌动蛋白的细胞骨架调节通路(P0.05)。3D vs Ti和Ti vs 2D差异甲基化基因在跨膜运输过程、Ca2+结合、细胞因子及受体作用通路等有差异显著性富集(P0.05),但3D vs 2D无差异。结论:1、肿瘤细胞在不同培养方法中表现的细胞生物学行为不同。2、MDA-MB-231细胞经二维、三维细胞培养和小鼠肿瘤细胞原位移植三种方法培养后其DNA甲基化状态存在差异,可能与其所在的生长环境相关。3、三维细胞培养模型在研究细胞间相互作用及细胞极性等生物学行为相比于二维细胞培养和肿瘤细胞原位移植模型有一定优势。
[Abstract]:Background and objective: breast cancer is China the highest incidence of female breast cancer tumors. Chinese annual number of new hair accounted for 12.2% of the world; the number of deaths accounted for 9.6%, after lung cancer, gastric cancer, liver cancer, esophageal cancer, colorectal cancer, the sixth leading cause of cancer deaths in women. Chinese cancer is multifactorial and induced in vivo in the complex environment in the body gradually evolved. That the accumulation of gene mutation is the main cause of cancer, but with the apparent paper and a growing number of genetics showed abnormal expression of genetic and epigenetic changes can cause gene. Epigenetic changes and progress of tumor research is very important. Show different cell culture method for the biological behavior of tumor cells under different performance, and the cells can increase or decrease the promoter methylation of gene function and effect of sub region The control of cell traits and malignant degree. But whether the change of DNA methylation by the method of cell culture is not clear. This study constructed 2D, 3D cell culture and tumor orthotopic transplantation model, observation of three kinds of culture differences in the growth of tumor cells model, explore the different growth environment to create a model of MDAMB-231 gene methylation status and related pathways differences. Through the results of this study further discusses the influence of gene methylation on tumor progression. Methods: to construct two-dimensional (two dimensional 2D) is a traditional training plane, three-dimensional (three dimensional 3D) cell culture and mouse orthotopic transplantation model (mouse orthotopic transplantation model, Tissue, Ti), using three kinds of model of cultured MDA-MB-231 cells, observe the growth of tumor cells in three models, and collecting cells using gene Group DNA Extraction Kit extraction, calculation and analysis of breast cancer MDA-MB-231 cell genome methylation sites of each gene in each application of DNA methylation of 450K chip and genomestudio software, respectively with Diff beta value, Score value, Delta_Beta said. The comparison of two kinds of training methods of breast cancer MDA-MB-231 cells genome methylation screening differentially methylated genes. And the use of online DAVID analysis tool for gene ontology on the theory analysis and analysis of cellular pathways. Results: 1, breast cancer cell MDA-MB-231 two-dimensional cell culture monolayer growth, cells were spindle shaped, the gap between the cell and cell connections exist only. Three dimensional cell culture of breast cancer MDA-MB-231 cells, a star shaped growth. Formed by a single cell cloning mass, there is a connection between cells and between cells and extracellular matrix. The mice orthotopic transplantation of MDA-MB-231 cells in mice The subcutaneous fat pad site grew a tumor mass, tumor tissue characters similar to the real.2, respectively after MDA-MB-231 tumor cells in 2D and 3D cell cultures were found 480 methylation differential gene (P0.05). MDA-MB-231 cell transplantation model between three-dimensional culture and in situ tumor was found in 86448 different genes (P0.05), there were 90005 different genes and tumor orthotopic transplantation model between MDA-MB-231 cells and cell culture of two-dimensional (P0.05).3, 3D cells cultured with two-dimensional cells cultured with tumor orthotopic transplantation model of three-dimensional cells, differences between gene methylation in the gene ontology part items have the same enrichment tumor orthotopic transplantation model and 2D cells are multicellular organism development (P0.05) and cell differentiation (P0.05) enrichment. Three differentially methylated genes in the cellular pathway model There are also some overlap, such as MAPK (P0.05) signaling pathway, cell adhesion molecules (P0.05) pathway, actin cytoskeleton pathway (P0.05).3D vs Ti and Ti vs differential methylation of 2D gene in process of transmembrane transport of Ca2+ binding, cytokine and receptor pathways for significant enrichment (P0.05), but no difference in 3D vs 2D. Conclusion: 1. The different.2 cell biological behavior of tumor cells in different culture methods, MDA-MB-231 cells by two-dimensional, three-dimensional cell culture and mouse tumor cells in situ transplantation with three kinds of method to raise the DNA methylation differences may be related to the growth environment.3, three-dimensional cell culture model in the study of interaction between cells and cell polarity and other biological behavior compared to two-dimensional cell culture and tumor cells in situ transplantation model has certain advantages.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9

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