貝塔類澱粉蛋白質對內皮細胞去氧核醣核酸甲基化之影響

The Effect of β-amyloid protein on DNA Methylation in Endothelial Cells

陳維捷(Wei-Chieh Chen)

指導教授 ： 王勝仕

共同指導教授 ： 劉懷勝(Hwai-Shen Liu)

國立臺灣大學/工學院/化學工程學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02342

摘要

DNA 甲基化之異常與許多疾病的病因息息相關，其中包括阿茲海默症(Alzheimer’s disease，AD) 。在阿茲海默症的發病機制中，類澱粉β蛋白質(β-amyloid，Aβ)是十分重要的因素；研究顯示，類澱粉β蛋白質在發病過程中會轉換成富含β-sheet的構形，並形成纖維狀(fibril)的結構，進而聚集成大量的糾結體(aggregate) 並造成細胞死亡。
文獻顯示，阿茲海默症與DNA甲基化有一定程度的關係，例如，阿茲海默症的病人其總體甲基化程度偏低、同型半胱氨酸(homocysteine,HCY)的濃度偏高等；此外，早衰性蛋白-1基因(presenilin-1,PS1)的去甲基化(demethylation)會造成類澱粉β蛋白質的加速產生。此些研究都指出甲基化之異常與阿茲海默症間有一定程度之關聯，然而其二者間真正之作用機制為何，至目前仍無定論。
本研究假設：類澱粉β纖維的毒性機制會對細胞DNA的甲基化產生影響。為測試此假設，我們使老鼠內皮細胞與不同濃度之類澱粉β纖維培養48小時後抽取細胞之DNA，將DNA以酵素水解後使用高效能液相層析儀(High performance liquid chromatography,HPLC)測量2'-脫氧胞2‘-脫氣胞普（ 5-Methyl-2 ’-deoxycytidine)的濃度，進而得到總體甲基化程度的情況。在實驗結果中我們發現，添加類澱粉β纖維會使總體甲基化程度下降，我們推測此可能由於類澱粉β蛋白質會造成細胞膜外的流動性下降，進而使甲基化轉移酶的活性下降；然而，此一推測還需進一步的實驗來證實。儘管如此，我們證實了類澱粉β纖維的確會對DNA甲基化機制產生影響並能造成甲基化程度下降的現象，我們希望此一成果相信對於類澱粉β纖維毒性機制的研究，以及類澱粉β蛋白質在阿茲海默症所扮演角色方面能有更進一步的認識。

關鍵字

DNA甲基化,內皮細胞,貝塔類澱粉蛋白質

並列摘要

The abnormality of DNA methylation has been found to be highly associated with many diseases including Alzheimer’s disease. β-amyloid peptide in Alzheimer’s disease plays a pivotal role in the pathogenesis of the disease. Researches showed that β-amyloid peptide self-assembles into stable fibrils with a characteristic cross β-pleated sheet secondary structure during the development of Alzheimer’s disease. Moreover, extensive protein fibrillogenesis or aggregation often occurs resulting in an accumulation of the abnormally folded proteins that damages cells.
Results from previous studies suggested that there exists a certain link between Alzheimer’s disease and DNA methylation. A lower level of total DNA methylation but a higher concentration of homocysteine was found in the patients with Alzheimer’s disease. In addition, the demthylation of presenilin-1 would induce a rapid production in β-amyloid peptides. While a correlation between the aberration of DNA methylation and Alzheimer’s disease pathology has been widely recognized, the detailed interaction mechanism remains rather elusive.
In the study presented here, we propose a hypothesis that β-amyloid peptide in an aggregation state would impact the DNA methylation. In order to test this hypothesis, the murine cerebral endothelial cells (MCEC) were first incubated with various concentrations of aggregated β-amyloid species for 48 hours and the extraction and enzymatic digestion of DNA from these cells were then performed. Via high performance liquid chromatography, the level of total DNA methylation was evaluated by the analysis of the concentrations of 2’-deoxycytidine and 5-methyl-2’-deoxycytidine. Our experimental results indicated that β-amyloid fibrils led to a reduction in the level of total DNA methylation in a concentration dependent manner. A possible explanation for the reduction in the level of DNA methylation is due to the β-amyloid-induced decline in the cell membrane fluidity which leads to a drop in the activity of methylatransferase, and therefore, a decrease in the level of total DNA methylation. However, further investigation is warranted to examine the underlying mechanism(s) of the interaction between

並列關鍵字

DNA mehtylation,Endothelial Cells,β-amyloid protein

參考文獻

33. 劉冠男. 類澱粉蛋白質與細胞膜間交互作用之探討. 台灣大學化學工程學研究所碩士論文 (2005).

1. Ahmad, I. & Rao, D. N. Chemistry and biology of DNA methyltransferases. Critical Reviews in Biochemistry and Molecular Biology 31, 361-380 (1996).

2. Razin, A. & Riggs, A. D. DNA Methylation and Gene-Function. Science 210, 604-610 (1980).

3. Freitag, M. & Selker, E. U. Controlling DNA methylation: many roads to one modification. Current Opinion in Genetics & Development 15, 191-199 (2005).

4. Chakrabarty, D., Yu, K. W. & Paek, K. Y. Detection of DNA methylation changes during somatic embryogenesis of Siberian ginseng (Eleuterococcus senticosus). Plant Science 165, 61-68 (2003).