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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊偉勛(Wei-Shiung Yang) | |
dc.contributor.author | Chih-Hsin Yang | en |
dc.contributor.author | 楊至心 | zh_TW |
dc.date.accessioned | 2021-05-17T09:15:23Z | - |
dc.date.available | 2017-09-18 | |
dc.date.available | 2021-05-17T09:15:23Z | - |
dc.date.copyright | 2012-09-18 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-10 | |
dc.identifier.citation | 參考文獻
1. Stirzaker, C. et al. Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors. Cancer Res 57, 2229-37 (1997). 2. Goldberg, A.D., Allis, C.D. & Bernstein, E. Epigenetics: a landscape takes shape. Cell 128, 635-8 (2007). 3. Costello, J.F. & Plass, C. Methylation matters. J Med Genet 38, 285-303 (2001). 4. Goll, M.G. & Bestor, T.H. Eukaryotic cytosine methyltransferases. Annu Rev Biochem 74, 481-514 (2005). 5. Smith, S.S., Kaplan, B.E., Sowers, L.C. & Newman, E.M. Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation. Proc Natl Acad Sci U S A 89, 4744-8 (1992). 6. Babinger, P., Volkl, R., Cakstina, I., Maftei, A. & Schmitt, R. Maintenance DNA methyltransferase (Met1) and silencing of CpG-methylated foreign DNA in Volvox carteri. Plant Mol Biol 63, 325-36 (2007). 7. Okano, M., Bell, D.W., Haber, D.A. & Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247-57 (1999). 8. Goll, M.G. et al. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science 311, 395-8 (2006). 9. Bonfils, C., Beaulieu, N., Chan, E., Cotton-Montpetit, J. & MacLeod, A.R. Characterization of the human DNA methyltransferase splice variant Dnmt1b. J Biol Chem 275, 10754-60 (2000). 10. Shock, L.S., Thakkar, P.V., Peterson, E.J., Moran, R.G. & Taylor, S.M. DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria. Proc Natl Acad Sci U S A 108, 3630-5 (2011). 11. Scarpulla, R.C. Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol Rev 88, 611-38 (2008). 12. Assaily, W. & Benchimol, S. Differential utilization of two ATP-generating pathways is regulated by p53. Cancer Cell 10, 4-6 (2006). 13. Wallace, D.C. & Fan, W. Energetics, epigenetics, mitochondrial genetics. Mitochondrion 10, 12-31 (2010). 14. Anderson, S. et al. Sequence and organization of the human mitochondrial genome. Nature 290, 457-65 (1981). 15. Ryan, M.T. & Hoogenraad, N.J. Mitochondrial-nuclear communications. Annu Rev Biochem 76, 701-22 (2007). 16. Wallace, D.C. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39, 359-407 (2005). 17. Taylor, R.W. & Turnbull, D.M. Mitochondrial DNA mutations in human disease. Nat Rev Genet 6, 389-402 (2005). 18. Rebelo, A.P., Williams, S.L. & Moraes, C.T. In vivo methylation of mtDNA reveals the dynamics of protein-mtDNA interactions. Nucleic Acids Res 37, 6701-15 (2009). 19. Shmookler Reis, R.J. & Goldstein, S. Mitochondrial DNA in mortal and immortal human cells. Genome number, integrity, and methylation. J Biol Chem 258, 9078-85 (1983). 20. Lee, H.C. & Wei, Y.H. Oxidative stress, mitochondrial DNA mutation, and apoptosis in aging. Exp Biol Med (Maywood) 232, 592-606 (2007). 21. Ricci, C. et al. Mitochondrial DNA damage triggers mitochondrial-superoxide generation and apoptosis. Am J Physiol Cell Physiol 294, C413-22 (2008). 22. Robertson, K.D. & Jones, P.A. DNA methylation: past, present and future directions. Carcinogenesis 21, 461-7 (2000). 23. Lengauer, C., Kinzler, K.W. & Vogelstein, B. DNA methylation and genetic instability in colorectal cancer cells. Proc Natl Acad Sci U S A 94, 2545-50 (1997). 24. Fernandez-Silva, P., Enriquez, J.A. & Montoya, J. Replication and transcription of mammalian mitochondrial DNA. Exp Physiol 88, 41-56 (2003). 25. Razin, A. & Cedar, H. DNA methylation and gene expression. Microbiol Rev 55, 451-8 (1991). 26. Ye, G. et al. Catalase protects cardiomyocyte function in models of type 1 and type 2 diabetes. Diabetes 53, 1336-43 (2004). 27. Kang, K.A., Zhang, R., Kim, G.Y., Bae, S.C. & Hyun, J.W. Epigenetic changes induced by oxidative stress in colorectal cancer cells: methylation of tumor suppressor RUNX3. Tumour Biol 33, 403-12 (2012). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6622 | - |
dc.description.abstract | 在近代生物遺傳學中發現了一種在不改變DNA序列的狀況下,造成表現型(phenotype) 的改變,並且可以繼續遺傳到子代的新興學問,稱為上位遺傳學(Epigenetics),其包含了DNA甲基化(DNA methylation)、組蛋白修飾(histone modification)、非編碼RNA(non-coding RNA)等。其中DNA甲基化被認為與基因印痕(gene imprinting)、胚胎發展(Embryonic development)、X染色體失活(X-chromosome inactivation)、癌化(oncogenesis)1與基因沉默(gene silencing)有關。而有關於細胞genomic DNA甲基化的研究更是十分的熱門,但卻對於細胞中另一個含有DNA及主要能量生產來源之胞器:「粒線體」的甲基化卻是少有研究。曾有前人研究指出,粒線體DNA的甲基化程度很低,且粒線體DNA甲基化與細胞存活度的關聯也被認為相當微弱。但在2009與2011年分別有文獻證明了粒線體DNA各個區域間有不同的可被甲基化程度與專屬粒線體的DNA甲基轉移酶。且近期由於偵測技術進步,我們認為粒線體的甲基化程度或許也應被重新評估。本研究將bisulfite conversion後的粒線體DNA利用焦磷酸定序法(Pyrosequecing)進行直接且更精確的甲基化偵測,並比較 HeLa 與 PC9 兩種細胞在H2O2所造成的氧化壓力下,粒線體的ATP6、ND1、ND6、TERM以及D-loop 等5個區域甲基化程度的變化。實驗結果顯示HeLa 與PC9在H2O2處理後,PC9顯現出較高的存活率,表示其對於氧化壓力具有較好的抗性與耐受性。在HeLa cells處理H2O2後,各基因甲基化程度並無顯著變化,但TERM與D –loop趨勢呈現上升;PC9 cells在H2O2處理後,5個區域的甲基化程度均是上升的。另一方面,在未處理H2O2的狀態下,HeLa cells甲基化程度皆高於PC9 cells,但經過H2O2處理後則為PC9 cells 較高且差異大。由本次研究結果我們推測,當細胞受到氧化壓力的影響時,會引起粒線體DNA的甲基化,並且不同細胞會有不同程度的差異。另外,我們也推測甲基化的程度會影響細胞在抵抗氧化壓力時的細胞存活率。 | zh_TW |
dc.description.abstract | Epigenetics which is defined as ” A phenomenon that changes the phenotype without changing the underlying DNA sequence, and it can inherit to offspring.” is one of the major research topics in modern biology. In addition, epigenetics includes DNA methylation, histone modification, and non-coding RNA. In which DNA methylation is associated with gene imprinting, embryonic development, X-chromosome inactivation, oncogenesis, and gene silencing. By focusing on DNA methylation, there are lots of researches in nuclear DNA, however, the information about mitochondrial DNA methylation is relatively rare. Nevertheless, it is known that mitochondria play an important role in ATP producing.
Previous study shows a low methylation pattern in mitochondria and consider that the relationship between mtDNA methylation and cell viability is weak . But, there were two papers which were published in 2009 and 2011 respectively were focusing on this issue as well. One indicates the different methylated accessibility of mtDNA and the other shows the appearance of mitochondrial DNA methyltransferase 1. Recently, the mitochondrial DNA methylation might need re-evaluation due to the detection technology progress. In this project, one of the major technology, pyrosequencing which is more direct and accurate method to detect oxisative stress-induced mtDNA methylation on HeLa cell and PC9 cell, is applied. By carrying out this technique, five mitochondrial-encode gene: ATP6, ND1, ND6, TERM and D-loop are analysed. Based on our research, PC9 cell has better viability than HeLa cell after H2O2 treatment. Moreover, methylation level of five genes are all increasing in PC9 cell, but no significant difference in HeLa cell. Therefore, baseline data shows that HeLa cell has higher methylation level than PC9 in mtDNA, however, the methylation level of PC9 cell is above than HeLa cell after H2O2 treatment. It is believed that oxidative stress-induced DNA methylation has cell-specificity and the methylation level might affect cell viability. | en |
dc.description.provenance | Made available in DSpace on 2021-05-17T09:15:23Z (GMT). No. of bitstreams: 1 ntu-101-P99448006-1.pdf: 1386754 bytes, checksum: 39aedecb5993343ad3d63772ebd82964 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 目錄
口委審定書.................................................................................................................... I 致謝............................................................................................................................... II 中文摘要...................................................................................................................... III Abstract ........................................................................................................................ IV 1. 緒論........................................................................................................................... 1 1.1 Epigenetics 與DNA 甲基化 ..................................................................... 1 1.2 DNMT ........................................................................................................ 2 1.3 粒線體........................................................................................................ 4 1.4 粒線體DNA 的甲基化需重新評估 ......................................................... 5 2. 材料與方法............................................................................................................... 7 2.1 細胞株培養................................................................................................ 7 2.2 Genomic DNA 萃取 ................................................................................. 7 2.3 RNA 萃取 ................................................................................................. 8 2.4 Reverse Transcription ................................................................................ 8 2.5 H2O2 處理 .................................................................................................. 8 2.6 MTS assay .................................................................................................. 9 2.7 qPCR .......................................................................................................... 9 2.8 Pyrosequencing .......................................................................................... 9 2.9 Bisulfite Conversion ................................................................................ 10 3. 結果......................................................................................................................... 11 3.1 Cell viability (H2O2) ................................................................................ 11 3.2 HeLa cell mtDNA 甲基化 ...................................................................... 11 3.3 PC9 cell mtDNA 甲基化 ........................................................................ 12 3.4 DNMT 1 expression ................................................................................. 13 4. 討論...................................................................................................................... 14 4.1 氧化壓力引發的粒線體DNA 甲基化會影響細胞存活率 ................... 14 4.2 粒線體DNA 甲基化具有基因與細胞的特異性 ................................... 15 4.3 D-loop 與ND6 高度甲基化對細胞的影響 ........................................... 16 4.4 H2O2 引發甲基轉移酶表現量與甲基化程度的改變 ............................ 17 4.5 檢討與未來展望...................................................................................... 17 參考文獻...................................................................................................................... 19 圖目錄 圖一、PC9 cell 經過H2O2 處理後的細胞存活率 ..................................................... 21 圖二、HeLa cell 經過H2O2 處理後的細胞存活率 ................................................... 22 圖三、HeLa 細胞ATP6 區域內所有CpG 位點甲基化之平均值 ........................... 23 圖四、HeLa 細胞ND1 區域內所有CpG 位點甲基化之平均值 ............................ 24 圖五、HeLa 細胞D-loop 區域內所有CpG 位點甲基化之平均值 ......................... 25 圖六、HeLa 細胞ND6 區域內所有CpG 位點甲基化之平均值 ............................ 26 圖七、HeLa 細胞TERM 區域內所有CpG 位點甲基化之平均值 ......................... 27 圖八、PC9 細胞ATP6 區域內所有CpG 位點甲基化之平均值 ............................. 28 圖九、PC9 細胞ND1 區域內所有CpG 位點甲基化之平均值 .............................. 29 圖十、PC9 細胞D-loop 區域內所有CpG 位點甲基化之平均值 ........................... 30 圖十一、PC9 細胞ND6 區域內所有CpG 位點甲基化之平均值 .......................... 31 圖十二、PC9 細胞TERM 區域內所有CpG 位點甲基化之平均值 ....................... 32 圖十三、甲基轉移酶引子示意圖.............................................................................. 33 圖十四、HeLa 與 PC9 total DNMT1 之表現量 ...................................................... 34 圖十五、HeLa 與 PC9 mtDNMT1 之表現量 .......................................................... 35 表目錄 表一、HeLa cell 甲基化趨勢統計 ............................................................................. 36 表二、PC9 cell 甲基化趨勢統計 ............................................................................... 37 表三、比較未處理H2O2 之HeLa cell 與PC9 cell 甲基化的差異 ......................... 38 表四、比較處理H2O2 後HeLa cell 與PC9 cell 甲基化的差異 ............................. 39 表五、交叉比較HeLa 與PC9 在H2O2 處理前後甲基化改變 ................................ 40 | |
dc.language.iso | zh-TW | |
dc.title | 氧化壓力引發的粒線體DNA甲基化對細胞存活率之影響 | zh_TW |
dc.title | Oxidative Stress-Induced Mitochondrial DNA Methylation Affect Cell Viability | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 吳君泰(June-Tai Wu) | |
dc.contributor.oralexamcommittee | 陳沛隆(Pei-Lung Chen) | |
dc.subject.keyword | 粒線體,甲基轉移酶,甲基化,氧化壓力, | zh_TW |
dc.subject.keyword | mitochondrial DNA,methyltransferase,methylation,oxidative stress, | en |
dc.relation.page | 40 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2012-08-10 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
顯示於系所單位: | 分子醫學研究所 |
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