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研究生:林冠豪
研究生(外文):Lin Guan-Hao
論文名稱:基因體規模肝細胞代謝網路之通量耦合與沃伯格效應分析
論文名稱(外文):Analysis of Fluxes Coupling and Warburg Effect on Genome-Scale Human Hepatocyte Network
指導教授:王逢盛
指導教授(外文):Wang Feng-Sheng
口試委員:周宜雄黃奇英鄒安平
口試委員(外文):Chou Yi-ShyongHuang Chi-YingTsou Ann-Ping
口試日期:2014-07-03
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:96
中文關鍵詞:最適化模擬肝臟肝細胞Mir122a 基因代謝網路膽紅素
外文關鍵詞:OptimizationLiverHepatocyteMir-122ametabolic networkBilirubin
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肝臟是身體內以代謝功能為主的器官,其中肝細胞 (Hepatocyte) 在身體裡面扮演著去毒素、儲存醣原(肝醣)和分泌蛋白質合成等重要生理機制,為人體重要的代謝器官。大部分的肝臟疾病都會有黃疸症狀,這是由於肝臟無法持續將膽紅素排出,所以就在體內累積而造成疾病。另外,粒線體 (Mitochondria) 為肝細胞經三羧酸循環 (Tricarboxylic acid cycle) 與呼吸鏈生成三磷酸腺苷 (Adenosine triphosphate,ATP) 之主要場所,提供細胞所需之能量,以維持肝細胞正常生理運作,但在異常細胞或癌細胞中,糖酵解 (glycolysis) 路徑通量會增強而粒線體裡面的氧化磷酸化(Oxidative phosphorylation)與三羧酸循環通量會減少。符合這種現象的狀態稱做沃伯格效應 (Warburg Effect)。
本研究利用人類肝細胞代謝網路模型- Recon2 liver hepatocytes model,藉由觀察以合成ATP反應之間的耦合反應關係,模擬肝細胞在攝取不同營養基質 (氧氣、胺基酸、脂肪酸、維生素) 的情況下,如何執行膽紅素 (Bilirubin) 排出等生理功能。並且假設人類肝細胞在受到環境干擾或基因突變等狀態下,會傾向於合成ATP通量最大化,以提供應變所需的能量,並且使用通量均衡分析方法 (Flux balance analysis, FBA) 求得在穩定狀態下的通量分佈。另外,我們以腫瘤抑制基因 Mir122a 為例,模擬 Mir122a 基因異常肝細胞的代謝變化,以突變通量均衡分析方法 (Mutant Flux balance analysis, mFBA) 求得在穩定狀態下的通量分佈,進行通量均衡分析與突變通量均衡分析的比較,最後再利用通量變異性分析 (Flux Variability Analysis, FVA) 發現其結果符合沃伯格效應並將模擬結果與實驗數據做交叉驗證,發現實驗組的22個重要物質裡面有14個物質模擬出來的結果與實驗相符。

Liver is a vital organ with the main metabolic functions in human body. The liver cells (Hepatocyte) inside the human body play a role of important physiological mechanism, including getting rid of the toxin, storage of glycogen and synthesis of secreted protein. Most of liver diseases occurred with jaundice symptoms, because bilirubin was unable discharged continuously by liver and accumulated in human body. Mitochondrion is the main organelle in which ATP (Adenosine Triphosphate) was generated through TCA cycle (Tricarboxylic Acid Cycle) and respiratory chain and provided hepatocyte cells enough energy to maintain their normal physiological operation. Unlike in normal cells, glycolysis is enhanced and both tricarboxylic acid cycle and oxidative phosphorylation capacity are reduced in various cancer cells. This phenomenon is corresponding to the Warburg Effect.
In this study, we used a mathematical hepatocyte-specific model downloaded from Recon2 to simulate bilirubin metabolic functions of liver under different environment conditions by observing the relationship of coupling between the objective function, e.g., different uptakes of oxygen, amino acids, lipids, and vitamins. We assumed mammalian hepatocyte cells will produce more ATP as possible as they can to meet the energy requirement for responding to genetic perturbations and environmental changes. The internal flux distribution of mutants at steady states can be obtained by flux balance analysis (FBA) under the assumption of maximization of internal fluxes. Mir122a is a tumor suppressor gene. We computed the flux distribution of a Mir122a mutant at steady states by mutant flux balance analysis (mFBA) and compare flux balance analysis and mutation flux balance analysis, found that the results is corresponding to the Warburg effect. Therefore, the simulation results and experimental data do cross-validation, found that 22 important substances which has 14 substances in agreement with experiment.

誌謝 I
摘要 II
Abstract IV
目錄 VI
圖目錄 IX
表目錄 XI
第一章 緒論 13
1.1 前言 13
1.2文獻回顧 16
1.3 研究動機 17
1.4 組織章節 19
第二章生物資料庫及工具程式簡介 20
2.1 引言 20
2.2 生物資料庫簡介 21
2.2-1 Kyoto Encyclopedia of Genes and Genomes (KEGG) 21
2.3工具程式簡介 24
第三章 計算方法的介紹及分析 29
3.1引言 29
3.2肝細胞代謝網路模式 30
3.3計算方法描述 32
第四章人體肝細胞代謝網路分析結果 41
4.1 引言 41
4.2 剔除腫瘤抑制基因Mir122的分析結果 42
4.2-1 Recon2 liver hepatocyte的模型規格 42
4.2-2生理功能目標設定 (Targets setting) 45
4.2-3正常細胞與剃除Mir122之結果分析 47
4.3通量耦合分析結果 56
4.4 剔除攝取反應的結果 57
4.5 比較正常細胞與不分泌linoelaidic acid反應及剃除GPT酵素結果 66
第五章 結論與未來展望 79
5.1結論 79
5.2 未來展望與建議 80
文獻回顧 81
附錄A 84
附錄B 90

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