臺灣博碩士論文加值系統

肥胖症為能量平衡失調的疾病，更是開發國家最常見之營養過度症。此外，肥胖也與糖尿病、高血壓、高血脂、心血管疾病、癌症等健康問題有關聯。然而，導致肥胖的脂肪代謝缺陷機轉極為複雜。因此，深入研究體內脂肪合成機轉為瞭解肥胖症病理過程之重大關鍵。 在肝臟或脂肪體中，脂肪酸合成酶(fatty acid synthase, FAS)利用acetyl-CoA、malonyl-CoA及NADPH來合成長鏈脂肪酸(C16或C18)，長鏈脂肪酸再轉變成三酸甘油酯(triglyceride)而貯存為脂肪。哺乳期間，硫酯酶II (thioesterase II, TE II)在乳腺表現並與FAS作用產生乳液內的中鏈脂肪酸(主要為C10)，能提供新生幼兒免疫保護功能及能量。研究指出TE II使FAS催化的脂肪酸合成反應提前中斷而產生中鏈脂肪酸。中鏈脂肪酸可直接進入粒線體進行β-氧化產生ATP，而能更有效提供細胞能量。由於TE II基因受嚴格調控通常不表現，我們採用基因傳送技術，在脂肪酸合成活躍但不具有TE II表現的肝細胞中使TE II表現，以進行其體內效應研究。據推測TE II表現會導致ATP生成增加，而同時減少長鏈脂肪酸的合成，進而減少脂肪的產生，所以可以應用於治療肥胖及肥胖相關之疾病。由於腺病毒基因傳送系統有多寄主性、高效價與低細胞毒性的優點。故在本實驗中，我們利用PCR的技術製作出重組的腺病毒載體Ad-TE II，並以西方點墨法與酵素分析法加以確認；以Ad-GFP作為指標，決定Ad-TE II感染HepG2細胞的最佳multiplicity of infection（MOI）為100~200；以西方點墨法及TE II酵素分析法確認以腺病毒傳送至HepG2細胞中之TE II基因確實有表現並具有酵素功能；以型態學觀察及細胞數目測定，發現腺病毒傳送TE II至肝細胞中表現後，會產生細胞毒性；經由生化學分析發現腺病毒傳送TE II至肝細胞中表現後，會增加肝臟細胞中的游離脂肪酸含量及天門冬胺酸轉胺酶和乳酸脫氫酶之釋出；以ATP luciferase assay發現腺病毒傳送TE II至肝細胞中表現後，會增加肝細胞中ATP的生成。綜合以上之研究結果，證明TE II傳送至肝細胞中表現後會引起細胞數目減少及損傷、游離脂肪酸增加、粒線體活性增加及ATP生成增加等現象。未來將繼續運用腺病毒基因傳送系統來探討TE II對於細胞的脂肪代謝。希望此研究不僅提供對於哺乳類動物細胞脂肪代謝更進一步的了解，也可用以評估TE II用於治療肥胖及其相關疾病之潛力。.

Obesity is a disorder of energy imbalance and the most prevalent nutritional diseases in developed countries. Besides, obesity is also strongly associated with health problems such as type 2 diabetes (NIDDM), hypertension, hyperlipidaemia, cardiovascular diseases and cancers. However, the defects in lipid metabolism underlying obesity-related disorders are extremely complicated. Thus, extensive studies on the mechanism of endogenous fatty acids synthesis would be one of the keys to elucidate molecular pathogenesis of obesity. In liver or adipose, fatty acid synthase (FAS) utilizes acetyl-CoA, malonyl-CoA and NADPH to synthesize long-chain fatty acids (C16 or C18), which can be converted to triglycerides and stored as fat. During lactation, thioesterase II (TE II) expresses in mammary glands and interacts with FAS to produce medium-chain fatty acid (primarily C10) in milk, which provides immune protection and energy for the newborn. TE II causes premature termination of fatty acid synthesis catalyzed by FAS and releases medium-chain fatty acids. Unlike long-chain fatty acids, medium-chain fatty acids can enter mitochondria directly for beta-oxidation to generate ATP, thus provide energy more efficiently. Since TE II gene expression is under strict regulation, we utilized adenovirus gene transfer techniques to deliver and express TE II in hepatocytes. It was postulated that expression of TE II in hepatocytes might result in the increase of ATP and reduction of long-chain fatty acids, subsequently decrease the fat production. Recombinant adenovirus was used as gene delivery system for TE II because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant Ad-TE II by PCR, western blot analysis, and enzymatic assay, respectively. By using Ad-GFP, we have determined the optimal multiplicity of infection (MOI) for adenovirus to infect HepG2 cells is about 100-200. Adenovirus-mediated TE II expression in hepatocytes was demonstrated by western blot as well as TE II enzymatic assay. We have demonstrated that the adenovirus-mediated TE II expression was slightly cytotoxic to hepatocytes. Besides, an increase of free fatty acids, asparate transaminase, lactate dehydrogenase levels, as well as ATP synthesis was also noted in the TE II-expressed hepatocytes. The enhanced the release of asparate transaminase (AST/GOT) and lactate dehydrogenase (LDH) after TE II expression in the hepatocytes further supported its cytotoxcity to hepatocytes. In the future, we will carry out experiments to further characterize the effects of TE II expression on cellular lipid metabolism through adenovirus gene delivery. We hope that the present studies will not only provide further insights into mammalian lipid metabolism, but also enable us to evaluate the therapeutic potential of TE II on the treatment of obesity and its related disorders.

目錄 頁次
摘要 6
關鍵詞 7
英文摘要 8
緒言 10
背景及重要性 10
脂肪酸合成概述 10
TE I釋放長鏈脂肪酸 13
TE II釋放中鏈脂肪酸 15
以TE II治療肥胖之潛力 17
腺病毒基因傳送治療 18
實驗設計及方法 21
細胞培養 21
量產及純化TE II重組腺病毒 22
以TE II重組腺病毒感染肝細胞 23
偵測TE II之表現 23
細胞數目之測定 23
細胞增殖分析試驗 24
ATP螢光素酶試驗 24
測定天門冬胺酸轉胺酶 25
測定乳酸脫氫酶 25
測定三酸甘油酯 26
分析肝細胞分泌之游離脂肪酸 26
肝細胞之生化指標篩選 27
統計分析 28
結果 29
製作並確認TE II重組腺病毒 29
偵測TE II之活性 29
測定腺病毒感染肝細胞之最適感染條件 29
偵測Ad-TE II在HepG2之表現 30
不同條件之培養基對表現TE II基因的肝細胞之影響 30
TE II基因傳送對肝細胞之影響 31
肝細胞之生化指標篩選 31
分析肝細胞分泌之FFA、GOT及LDH 32
肝細胞三酸甘油酯分析 33
討論 34
參考文獻 41

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