Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/99406
標題: 葉酸代謝基因變化影響胞內代謝路徑平衡之初步探討
Folate metabolic gene variations affect cellular metabolic kinetics-a pilot study
作者: 葉韋廷
Wei-Ting Ye
關鍵字: RNA干擾;細胞模式;葉酸代謝;代謝流追蹤;碳源分配;RNA interference;folate metabolism;glycolysis;tricarboxylic acid cycle;fatty acid synthesis;amino acid metabolism
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摘要: 
背景及目的 本研究針對葉酸代謝基因變化影響胞內代謝路徑平衡之初步探討。在葉酸代謝單碳循環中許多關鍵酵素扮演重要角色。例如亞甲基四氫葉酸還原酶負責催化將5,10-甲基四氫葉酸轉換為5-甲基四氫葉酸,此不可逆反應為連結甲基循環與葉酸循環間之重要橋樑。歸功於次世代定序技術逐年普及發達,愈來愈多葉酸代謝基因之基因多態性已經被陸續發現。流行病學研究顯示包含亞甲基四氫葉酸還原酶等葉酸之基因多態性影響多種人類疾病發生之風險或預後,包括一些重要的代謝相關疾病如心血管疾病、糖尿病及癌症等。因此帶有葉酸代謝單碳循環中關鍵酵素常見基因多態性如何影響代謝路徑調控已是人類疾病預防治療之重要議題。然而葉酸代謝單碳循環中許多關鍵酵素在單碳代謝上所扮演之角色雖已闡明,但其對於其它細胞內重要關鍵之代謝路徑,包括醣解作用、檸檬酸循環、磷酸戊糖途徑、脂質代謝、胺基酸轉換及胞內碳源利用分配之調控目前尚未被系統性地探討。本論文開啟系列研究,設計實驗針對葉酸代謝單碳循環中關鍵酵素其可能在代謝路徑中扮演之角色開啟初步探討。
實驗設計 以本研究室先前以short hairpin RNA干擾技術所建立之穩定細胞株為模式,同時以 wildtype 及RNA干擾一非哺乳動物之基因作為negative control作為雙對照組 cell-lines進行研究。將不同細胞培養在正常、缺乏或是補充關鍵營養素之情況,觀察其生長情況,探討關鍵代謝酵素蛋白表現並根據不同研究目地以不同穩定性同位素進行標定,追蹤葉酸代謝單碳循環中關鍵酵素表現低下或是過度表現之細胞,其胞內特定代謝路徑如何受到該基因調控,並進一步探索當此關鍵基因表現異常時,在營養素受限情況下碳源如何進行分配利用。
結果及結論 我們發現特定葉酸代謝單碳循環關鍵酵素基因表達低下之細胞在碳源的選擇與wildtype細胞株及negative control對照細胞有很大差異。目標基因表達不僅影響葡萄糖碳源進入檸檬酸循環,亦控制蘋果酸形成用於合成天門冬胺酸並影響酮戊二酸的碳源用於合成麩胺酸。此外還會調節醋酸鹽碳源用於脂肪酸生合成,進而影響脂質代謝。總結來說,我們的初步研究發現特定葉酸代謝單碳循環關鍵酵素基因表達會直接影響細胞內主要代謝路徑其碳源之選擇一先順序及利用分配。本研究開啟了多個新穎研究方向,對於特定葉酸代謝單碳循環關鍵基因表達或是酵素功能如何影響細胞內代謝流向平衡有多向啟發,未來透過進一步設計實驗探討基因、營養素、藥物間的交互作用間之調控機制,其能對於葉酸代謝單碳循環關鍵基因功能異常或是基因多樣性的相關代謝疾病提供預防或是治療的新觀點。

Folate metabolic genes regulate folate-dependent methyl group homeostasis and nucleitide syntheses. However, the role of these genes in other essential metabolic pathways is unclear. Currently, no guidelines are established for routine pharmacogenomic testing involving folate metabolic genes in long-term metabolic disease therapies. We aimed to investigate the potential role of folate metabolic genes in key metabolic pathways.
Study design. Cell-ines with stabilized target folate metabolic gene inhibition using shRNA delivered by a Lentiviral vector were used Stable isotope labeling experiments were designed and performed in cells models to investigate how specific folate target genes modulate the carbon flow and the utilization of different carbon sources intracelluylarly.
Results and conclusions. In vitro, interference of one of the target folate gene expression altered glycolysis and the glucose carbon flow entering succinate, fumarate, malate, and ⍺-ketoglutarate. In addition, it changed the utilization of glucose for de novo amino acid synthesis and fatty acid syntheses from acetate.
The present study opens the door for folate metabolic genes in cellular essetntal metabolic pathways including glycolysis; tricarboxylic acid cycle; fatty acid synthesis; amino acid metabolism.
URI: http://hdl.handle.net/11455/99406
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