本文選題：腫瘤　切入點：DNA甲基化　出處：《吉林大學》2016年碩士論文

【摘要】：背景與目的:乳腺癌是中國女性發(fā)病率最高的腫瘤。每年中國乳腺癌新發(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,第六大中國女性癌癥死亡原因。腫瘤發(fā)生是體內(nèi)多因素誘導并在體內(nèi)的復雜環(huán)境逐步演化而成。認為基因突變的積累是腫瘤發(fā)生的主要原因,但隨著表觀遺傳學的提出和越來越多的研究顯示遺傳學和表觀遺傳學的改變均可引起基因的異常表達。表觀遺傳學的改變對腫瘤的發(fā)生和進展十分重要。研究顯示不同的細胞培養(yǎng)方法下腫瘤細胞的生物學行為表現(xiàn)也不同,且細胞能通過升高或降低基因啟動子區(qū)域的甲基化影響基因功能從而控制細胞的性狀和惡性度。但DNA甲基化狀態(tài)的改變是否受細胞培養(yǎng)方法的影響尚不清楚。本研究通過構建2D、3D細胞培養(yǎng)和腫瘤原位移植模型,觀察三種培養(yǎng)模型下腫瘤細胞的生長方式差別,探索不同模型所營造的生長環(huán)境對MDAMB-231細胞基因甲基化狀態(tài)及相關細胞通路差異性。通過本研究結果進一步論述基因甲基化狀態(tài)對腫瘤發(fā)生進展過程的影響。方法:構建二維(two dimensional,2D)即傳統(tǒng)平面培養(yǎng)、三維(three dimensional,3D)細胞培養(yǎng)和小鼠原位移植模型(mouse orthotopic transplantation model,簡稱Tissue,Ti),采用三種模型培養(yǎng)MDA-MB-231細胞,觀察三種模型下腫瘤細胞的生長方式,并收集細胞,使用基因組抽提試劑盒提取DNA,應用DNA甲基化450K芯片和genomestudio分析軟件計算分析乳腺癌MDA-MB-231細胞基因組每個基因每個甲基化位點,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細胞基因組甲基化狀態(tài)篩選差異的甲基化基因,并運用DAVID在線分析工具對其進行基因本體論分析和細胞通路分析。結果:1、二維細胞培養(yǎng)的乳腺癌MDA-MB-231細胞貼壁單層生長,細胞呈梭形,僅存在細胞與細胞之間的縫隙連接。三維細胞培養(yǎng)的乳腺癌MDA-MB-231細胞,呈星球狀生長,由單個細胞形成克隆團塊,存在細胞與細胞間及細胞與細胞外基質(zhì)間的連接。小鼠原位移植的MDA-MB-231細胞在小鼠皮下脂肪墊部位生長出一個腫瘤團塊,性狀類似真實的腫瘤組織。2、分別經(jīng)過二維和三維細胞培養(yǎng)后的MDA-MB-231腫瘤細胞查出480個甲基化狀態(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細胞則發(fā)現(xiàn)86448個有差異基因(P0.05),而二維細胞培養(yǎng)與腫瘤原位移植模型間的MDA-MB-231細胞有90005個差異基因(P0.05)。3、三維細胞培養(yǎng)與二維細胞培養(yǎng)、三維細胞培養(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,分別是多細胞生物體發(fā)育(P0.05)和細胞分化(P0.05)。三種模型間差異甲基化基因在細胞通路上的富集也存在部分重合,如MAPK信號通路(P0.05)、細胞黏附分子作用通路(P0.05)、肌動蛋白的細胞骨架調(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輸過程、Ca2+結合、細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無差異。結論:1、腫瘤細胞在不同培養(yǎng)方法中表現(xiàn)的細胞生物學行為不同。2、MDA-MB-231細胞經(jīng)二維、三維細胞培養(yǎng)和小鼠腫瘤細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環(huán)境相關。3、三維細胞培養(yǎng)模型在研究細胞間相互作用及細胞極性等生物學行為相比于二維細胞培養(yǎng)和腫瘤細胞原位移植模型有一定優(yōu)勢。
[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.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R737.9

【相似文獻】

相關期刊論文 前5條

1 周玉球;DNA甲基化分析技術的研究進展[J];國外醫(yī)學.遺傳學分冊;2001年06期

2 羅君;凌志強;;DNA甲基化分析技術的研究進展[J];國際檢驗醫(yī)學雜志;2013年06期

3 黃朝暉;李莉華;楊帆;劉志輝;胡瑜;宋明旭;任金冬;;糞便DNA甲基化分析在結直腸癌診斷中的應用價值[J];中華檢驗醫(yī)學雜志;2007年06期

4 趙貴森;鄭海燕;莫耀南;馮文芳;楊渝珍;;一種用于DNA甲基化分析的改進型亞硫酸氫鹽轉化法[J];中國生物化學與分子生物學報;2007年10期

5 ;[J];;年期

相關會議論文 前5條

1 何卓培;徐淑平;徐俊;熊守仁;蘇錦文;;亞毫米波輻照與水稻體細胞無性系變異后代的DNA甲基化分析[A];中國細胞生物學學會第五次會議論文摘要匯編[C];1992年

2 張園秀;徐榕青;林文津;郭娜;張亞敏;;基于MSAP的枇杷葉DNA甲基化分析[A];2014年全國中藥學術研討會暨中國中西醫(yī)結合學會第六屆中藥專業(yè)委員會換屆改選會論文集[C];2014年

3 侯睿;包振民;王師;王宸;李寧;王明玲;;中國沿海三種主要養(yǎng)殖扇貝的DNA甲基化分析[A];中國動物學會、中國海洋湖沼學會貝類學會分會第十四次學會研討會論文摘要匯編[C];2009年

4 于雪梅;江漢民;王春國;陳成彬;孫德嶺;宋文芹;;花椰菜DH群體中不同農(nóng)藝性狀的DNA甲基化分析[A];中國的遺傳學研究——遺傳學進步推動中國西部經(jīng)濟與社會發(fā)展——2011年中國遺傳學會大會論文摘要匯編[C];2011年

5 陳力;李秀蘭;趙磊;董風平;;藥用植物丹參的遺傳改良與種質(zhì)創(chuàng)新研究Ⅳ、三倍體丹參優(yōu)勢與DNA甲基化分析[A];中國植物學會七十五周年年會論文摘要匯編（1933-2008）[C];2008年

相關博士學位論文 前4條

1 孫晗;小麥光周期基因Photoperiod-B1的DNA甲基化分析[D];山東農(nóng)業(yè)大學;2013年

2 汪衛(wèi)星;天然與人工合成三倍體枇杷基因組變異及其DNA甲基化分析[D];西南大學;2008年

3 劉仲敏;食管鱗癌表達下調(diào)基因DNA甲基化分析及其相關功能的研究[D];中國協(xié)和醫(yī)科大學;2004年

4 魏華麗;落葉松體細胞胚胎發(fā)生過程中DNA甲基化分析及MET1、DDM1克隆研究[D];中國林業(yè)科學研究院;2010年

相關碩士學位論文 前7條

1 喬莎;乳腺癌MDA-MB-231細胞不同培養(yǎng)模型的構建及DNA甲基化分析[D];吉林大學;2016年

2 朱恒星;四倍體長春花（Catharanthus roseus）生物學特性研究及DNA甲基化分析[D];西南大學;2009年

3 劉洋洋;DNA甲基化分析中重亞硫酸鹽處理DNA樣本的轉化效率評估方法的研究[D];南京大學;2015年

4 何波;沙田柚（3x、4x）與紅江橙（4x）多倍體基因組變異及其DNA甲基化分析[D];西南大學;2009年

5 邊禹;不同倍性多倍體枇杷基因組變異及其DNA甲基化分析[D];西南大學;2010年

6 孫其愷;肝細胞癌TACE治療前后血漿中游離DNA甲基化分析及其臨床預后價值[D];安徽醫(yī)科大學;2014年

7 賈志剛;三倍體枇杷實生后代及雜交后代SCoT標記及DNA甲基化分析[D];西南大學;2011年

，

本文編號：1700377