發(fā)布時(shí)間：2018-04-02 12:20

  本文選題：腫瘤　切入點(diǎn)：DNA甲基化　出處：《吉林大學(xué)》2016年碩士論文

【摘要】：背景與目的:乳腺癌是中國(guó)女性發(fā)病率最高的腫瘤。每年中國(guó)乳腺癌新發(fā)數(shù)量占全世界的12.2%;死亡數(shù)量占9.6%,是繼肺癌、胃癌、肝癌、食管癌、大腸癌之后,第六大中國(guó)女性癌癥死亡原因。腫瘤發(fā)生是體內(nèi)多因素誘導(dǎo)并在體內(nèi)的復(fù)雜環(huán)境逐步演化而成。認(rèn)為基因突變的積累是腫瘤發(fā)生的主要原因,但隨著表觀遺傳學(xué)的提出和越來(lái)越多的研究顯示遺傳學(xué)和表觀遺傳學(xué)的改變均可引起基因的異常表達(dá)。表觀遺傳學(xué)的改變對(duì)腫瘤的發(fā)生和進(jìn)展十分重要。研究顯示不同的細(xì)胞培養(yǎng)方法下腫瘤細(xì)胞的生物學(xué)行為表現(xiàn)也不同,且細(xì)胞能通過(guò)升高或降低基因啟動(dòng)子區(qū)域的甲基化影響基因功能從而控制細(xì)胞的性狀和惡性度。但DNA甲基化狀態(tài)的改變是否受細(xì)胞培養(yǎng)方法的影響尚不清楚。本研究通過(guò)構(gòu)建2D、3D細(xì)胞培養(yǎng)和腫瘤原位移植模型,觀察三種培養(yǎng)模型下腫瘤細(xì)胞的生長(zhǎng)方式差別,探索不同模型所營(yíng)造的生長(zhǎng)環(huán)境對(duì)MDAMB-231細(xì)胞基因甲基化狀態(tài)及相關(guān)細(xì)胞通路差異性。通過(guò)本研究結(jié)果進(jìn)一步論述基因甲基化狀態(tài)對(duì)腫瘤發(fā)生進(jìn)展過(guò)程的影響。方法:構(gòu)建二維(two dimensional,2D)即傳統(tǒng)平面培養(yǎng)、三維(three dimensional,3D)細(xì)胞培養(yǎng)和小鼠原位移植模型(mouse orthotopic transplantation model,簡(jiǎn)稱Tissue,Ti),采用三種模型培養(yǎng)MDA-MB-231細(xì)胞,觀察三種模型下腫瘤細(xì)胞的生長(zhǎng)方式,并收集細(xì)胞,使用基因組抽提試劑盒提取DNA,應(yīng)用DNA甲基化450K芯片和genomestudio分析軟件計(jì)算分析乳腺癌MDA-MB-231細(xì)胞基因組每個(gè)基因每個(gè)甲基化位點(diǎn),分別用β值、Diff Score值、Delta_Beta表示。比較兩種培養(yǎng)方法間乳腺癌MDA-MB-231細(xì)胞基因組甲基化狀態(tài)篩選差異的甲基化基因,并運(yùn)用DAVID在線分析工具對(duì)其進(jìn)行基因本體論分析和細(xì)胞通路分析。結(jié)果:1、二維細(xì)胞培養(yǎng)的乳腺癌MDA-MB-231細(xì)胞貼壁單層生長(zhǎng),細(xì)胞呈梭形,僅存在細(xì)胞與細(xì)胞之間的縫隙連接。三維細(xì)胞培養(yǎng)的乳腺癌MDA-MB-231細(xì)胞,呈星球狀生長(zhǎng),由單個(gè)細(xì)胞形成克隆團(tuán)塊,存在細(xì)胞與細(xì)胞間及細(xì)胞與細(xì)胞外基質(zhì)間的連接。小鼠原位移植的MDA-MB-231細(xì)胞在小鼠皮下脂肪墊部位生長(zhǎng)出一個(gè)腫瘤團(tuán)塊,性狀類似真實(shí)的腫瘤組織。2、分別經(jīng)過(guò)二維和三維細(xì)胞培養(yǎng)后的MDA-MB-231腫瘤細(xì)胞查出480個(gè)甲基化狀態(tài)有差異的基因(P0.05)。三維培養(yǎng)和腫瘤原位移植模型間的MDA-MB-231細(xì)胞則發(fā)現(xiàn)86448個(gè)有差異基因(P0.05),而二維細(xì)胞培養(yǎng)與腫瘤原位移植模型間的MDA-MB-231細(xì)胞有90005個(gè)差異基因(P0.05)。3、三維細(xì)胞培養(yǎng)與二維細(xì)胞培養(yǎng)、三維細(xì)胞培養(yǎng)與腫瘤原位移植模型、腫瘤原位移植模型與二維細(xì)胞培養(yǎng)間的差異性甲基化基因在基因本體論部分條目上均有相同富集,分別是多細(xì)胞生物體發(fā)育(P0.05)和細(xì)胞分化(P0.05)。三種模型間差異甲基化基因在細(xì)胞通路上的富集也存在部分重合,如MAPK信號(hào)通路(P0.05)、細(xì)胞黏附分子作用通路(P0.05)、肌動(dòng)蛋白的細(xì)胞骨架調(diào)節(jié)通路(P0.05)。3D vs Ti和Ti vs 2D差異甲基化基因在跨膜運(yùn)輸過(guò)程、Ca2+結(jié)合、細(xì)胞因子及受體作用通路等有差異顯著性富集(P0.05),但3D vs 2D無(wú)差異。結(jié)論:1、腫瘤細(xì)胞在不同培養(yǎng)方法中表現(xiàn)的細(xì)胞生物學(xué)行為不同。2、MDA-MB-231細(xì)胞經(jīng)二維、三維細(xì)胞培養(yǎng)和小鼠腫瘤細(xì)胞原位移植三種方法培養(yǎng)后其DNA甲基化狀態(tài)存在差異,可能與其所在的生長(zhǎng)環(huán)境相關(guān)。3、三維細(xì)胞培養(yǎng)模型在研究細(xì)胞間相互作用及細(xì)胞極性等生物學(xué)行為相比于二維細(xì)胞培養(yǎng)和腫瘤細(xì)胞原位移植模型有一定優(yōu)勢(shì)。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9

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