臺灣博碩士論文加值系統

　　本研究利用實驗室先前發現的新型小分子藥物－ENERGI作為研究主體，根據其已知生化途徑並藉由人類細胞株HAP1細胞作為模式細胞以不同濃度及時間點偵測其下游標的單磷酸腺苷活化蛋白質激酶（AMP-activated protein kinase, AMPK）及乙醯輔酶A羧化酶（Acetyl-CoA carboxylase, ACC）之磷酸化及三磷酸腺苷（Adenosine triphosphate, ATP）變化，根據其ATP及AMPK磷酸化之結果，找出最佳藥物劑量後與具延長壽命相關潛力藥物維他命B3或α-酮戊二酸（α- ketoglutarate, AKG）併用。維他命B3為菸鹼醯胺腺嘌呤二核苷酸（Nicotinamide adenine dinucleotide, NAD+）前驅物，而NAD+是涉及許多生理過程的重要輔酶，包括代謝、抵抗自由基和去氧核醣核酸之修復，許多研究表明隨著年齡增長NAD+的含量大幅下降，也有研究顯示NAD+的補充可以延長多種模式生物的壽命，例如線蟲、果蠅、老鼠等。α-酮戊二酸為決定三羧酸循環總體速率的關鍵分子，最近的一項研究顯示AKG可透過抑制ATP合成酶和TOR來延長成年秀麗隱桿線蟲的壽命並延緩年齡相關的疾病。以此文獻作為研究基礎，本研究以秀麗隱桿線蟲（Caenorhabditis elegans）做為動物模型探討ENERGI是否能增加其壽命（lifespan）。HAP1細胞實驗結果顯示ENERGI可提升細胞內ATP含量，並可提升經α-酮戊二酸作用後下降的整體ATP含量。維他命B3隨濃度提升可提升ATP含量，但效力不及ENERGI，且此二藥物併用後，可提升NAD+含量，在利用秀麗隱桿線蟲做為動物模型的相關實驗中，ENERGI可有效增加其健康壽命（healthspan），但不能延長其整體壽命。

　　In this study, we used a novel small molecular drug, ENERGI, as the major treatment. By using the HAP1 cell line as the cell model, different concentrations and the times of treatment were investigated to find the fitness of drug dosage. Furthermore, we detected intracellular ATP level and the phosphorylated level of AMP-activated protein kinase (AMPK) to find the effective treatment of ENERGI for further combining treatment with Vitamin B3 or alpha-ketoglutarate. Vitamin B3 is the precursor of nicotinamide adenine dinucleotide (NAD+), an important coenzyme, which involved in numerous physiological processes, including metabolism, protection from free radical, and DNA repair. Many studies demonstrated that NAD+ level decreased with aging. In addition, many studies have demonstrated that increases in NAD+ level can extend the lifespan in various organisms. Alpha-ketoglutarate is a key molecule which determines the overall rate in the tricarboxylic acid cycle. Recently, a study showed that alpha-ketoglutarate could extend the lifespan of mature Caenorhabditis elegans (C. elegans) and delays age-related diseases by inhibiting ATP synthase. For these reasons, we used C. elegans as an animal model to find out whether ENERGI could extend the lifespan of C. elegans or not. The results from the experiments of HAP1 cells showed that ENERGI could increase intracellular ATP level, even if alpha-ketoglutarate decrease the ATP level. Except for ENERGI, Vitamin B3 can also increase ATP level but the effectiveness is lower than ENERGI. There is no doubt that co-treatment of ENERGI and vitamin B3 can increase NAD+ level. In C. elegans model, ENERGI can extends its healthspan but not lifespan.

目錄
中文摘要 6
英文摘要 7
一、概論 8
1.1 ENERGI 8
1.2 AMP – 活化蛋白質激酶(AMP-activated protein kinase, AMPK ) 9
1.2.1 AMPK基本結構 9
1.2.2 AMPK酵素調控機制 10
1.2.3已知AMPK與長壽相關機制 12
1.3 α – 酮戊二酸(α - ketoglutarate, AKG ) 12
1.3.1已知 AKG 與長壽相關機制 12
1.4 維他命B3 13
1.4.1菸鹼醯胺( Nicotinamide, NAM ) 13
1.4.2菸鹼酸( Nicotinic acid, NA ) 14
1.5 菸鹼醯胺腺嘌呤二核苷酸( Nicotinamide adenine dinucleotide, NAD+ ) 14
1.5.1已知NAD+與長壽相關機制 15
1.6 研究動機和目的 15
二、實驗結果 16
2.1確認ENERGI對HAP1細胞之最佳加藥反應時間 16
2.2確認ENERGI對HAP1細胞之有效加藥濃度 17
2.3確認ENERGI對HAP1細胞之最低有效濃度 17
2.4確認ENERGI和AKG併用對HAP1細胞之有效濃度 18
2.5確認ENERGI和NA併用對HAP1細胞之有效濃度 18
2.6確認ENERGI和NAM併用對HAP1細胞之有效濃度 18
2.7測試ENERGI是否可延長秀麗隱桿線蟲之壽命 19
2.8未來之研究方向 19
三、實驗討論 21
圖表集 25
圖一、不同時間ENERGI處理下HAP1細胞內AMPK磷酸化程度變化 25
圖二、不同濃度ENERGI處理下HAP1細胞內AMPK磷酸化程度變化及ATP含量 26
圖三、低濃度ENERGI處理下HAP1細胞內AMPK磷酸化程度變化及ATP含量 27
圖四、以ENERGI和不同濃度AKG共同處理後，HAP1細胞內AMPK磷酸化程度變化及其APRT剔除細胞中ATP含量 28
圖五、以ENERGI和不同濃度NA共同處理後測定HAP1細胞中ATP含量及NAD+ 含量 29
圖六、以ENERGI和不同濃度NAM共同處理後測定HAP1細胞中ATP含量及NAD+ 含量 30
圖七、以秀麗隱桿線蟲做為動物模型測試ENERGI是否可延長其壽命 31
四、材料與方法 32
4.1　貼附型細胞培養 32
4.2　細胞保存 34
4.3　細胞破裂法 36
4.4　蛋白質定量 38
4.5　電泳檢定法 40
4.6　蛋白質轉印法 43
4.7　酵素免疫染色法 45
4.8　ATP測試法 47
4.9　NAD+測試法 48
4.10　秀麗隱桿線蟲之培養 51
4.11　Escherischia coli OP50培養盤製作方式 52
4.12　Escherischia coli OP50培養方式 52
4.13　Carbenicillin (Carb)藥品配置 53
4.14　線蟲壽命追蹤實驗Lifespan Assay 53
五、參考資料 56

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