臺灣博碩士論文加值系統

老化是每種生物體的必經過程，但老化常常伴隨著老化相關疾病，例如阿茲海默症、第二型糖尿病、記憶力衰退等等。研究指出生物體老化與老化相關疾病的產生有關，已知的抗老化藥物也具有改善老化相關疾病的功效。尋找延緩老化相關藥物，也有機會用於老化相關疾病的治療。到目前為止，儘管人類不斷在尋找能夠延長壽命的物質，但影響人體老化的相關機制牽連著許多複雜要素，因此找到能夠延長壽命的藥物並不多。
本論文利用Mother enrichment program (MEP) 快篩系統，發現蓮心酒萃物可能具有延長酵母菌壽命之效果。接著以Replicative life span (RLS) analysis驗證蓮心酒萃物可以延長酵母菌RLS的效果，發現蓮心酒萃物經由Sir2-dependent路徑延長酵母菌RLS。我們進一步利用MEP analysis篩選從蓮心酒萃物分離出的純化物，發現到純化物PN6在MEP analysis中可以增加酵母菌壽命。利用RLS analysis再次驗證 PN6確實能夠增加酵母菌RLS。我們也發現 PN6是經由Sir2-dependent路徑延長酵母菌RLS。除此之外，我們也發現PN6的類似物PN6-A在RLS analysis也能夠增加酵母菌RLS，並經由Sir2-dependent路徑延長酵母菌RLS且發現蓮心酒萃物與PN6-A皆能顯著性提升SIRT1/Sir2活性，蓮心酒萃物還能夠顯著性提升細胞內的NAD+濃度。

Aging is a process that happens to most creature, and it is often accompanied with aging-related diseases. It is important to find aging-delaying drugs as it can be used in treatment of aging-related diseases. This study utilized rapid test system of mother enrichment program (MEP), and found that ethanolic extract of Plumula Nelumbinis may have lifespan extension effect in yeast. Furthermore, we used replicative lifespan analysis (RLS) to confirm the lifespan extension activity of ethanol extract of Plumula Nelumbinis, and observed that it extends replicative lifespan in yeast through Sir2-dependent pathway. Moreover, by MEP, we also found a compound eluded form Plumula Nelumbinis extract, PN6 has a potential ability to increase lifespan in yeast. Next, our RLS analysis demonstrated that PN6 elevates RLS in yeast through Sir2-dependent pathway. We also found that the analogs of PN6, PN6-A possessed great ability to extend lifespan in yeast through Sir2-dependent pathway ; ethanolic extract of Plumula Nelumbinis and PN6-A both significantly induced SIRT1/Sir2 activity-- ethanolic extract of Plumula Nelumbinis also could increase NAD+ level in vitro. Based on the above results, we concluded that PN6 and PN6-A have considerable potential for extending lifespan and treating aging-related diseases.

指導教授推薦書
口試委員審定書
誌謝 iii
中文摘要 iv
Abstract v
目錄 vi
圖目錄 ix
表目錄 xi
縮寫表 xii
第一章 簡介 1
1.1 研究動機 1
1.2 老化學說 1
1.3 酵母菌於老化研究上之優勢 3
1.4 Chronological life span (CLS) 和Replicative life span (RLS) 3
1.5 Mother enrichment program (MEP) 4
1.6 蓮心 (Plumula Nelumbinis ; PN) 4
1.6.1抗氧化活性 4
1.6.2抗高血壓活性 4
1.6.3抗氣喘活性 5
1.6.4抗冠狀動脈狹窄 (Restenosis) 的活性 5
1.6.5抑制神經退化性疾病的活性 5
1.6.6抗糖尿病的活性 5
1.6.7抑制自體免疫疾病的活性 5
1.6.8抗心律不整的活性 6
1.6.9抗阿茲海默症的活性 6
1.6.10抗癌活性 6
1.6.11保肝活性 6
1.6.12抗發炎活性 7
1.6.13抗老化活性 7
1.7 卡路里限制 (Calorie restriction ; CR) 7
1.8 Silent Information Regulator2 (SIR2) 8
1.9 Sirtuin1 (SIRT1) 9
1.10 Target of rapamycin (TOR) 9
第二章實驗材料與方法 11
2.1.1藥品 11
2.1.2抗體資料表 11
2.1.3酵母菌株 11
2.2酵母菌基因型資料表 11
2.3實驗方法 12
2.3.1 酵母菌培養 12
2.3.2 Yeast transformation 12
2.3.3 Liquid aging assay 13
2.3.4 Replicative life span analysis 13
2.3.5 Yeast TCA whole cell extracts 14
2.3.6 Western blot 14
2.3.7 Calculation of Intracellular NAD+ Content 15
2.3.8 SIRT1 in vitro activity assay 15
第三章 結果 16
3.1 蓮心酒萃物在MEP analysis中增加酵母菌壽命 16
3.2 蓮心酒萃物延長酵母菌的Replicative life span (RLS) 16
3.3 蓮心酒萃物經由Sir2-dependent路徑延長酵母菌RLS 17
3.4 蓮心酒萃物經由Tor-independent的路徑延長酵母菌RLS 18
3.5 蓮心酒萃物無法提升Sir2蛋白質表現量 18
3.6 蓮心酒萃物無法提升Pnc1蛋白質表現量 19
3.7 蓮心酒萃物提高SIRT1活性 19
3.7 蓮心酒萃物提高酵母菌NAD+濃度 19
3.8 PN6在MEP analysis中增加酵母菌壽命 20
3.9 PN6增加酵母菌RLS 21
3.10 PN6經由Sir2-dependent路徑延長酵母菌RLS 21
3.11 PN6經由TOR-independent路徑延長酵母菌RLS 21
3.12 PN6-A增加酵母菌RLS 22
3.13 PN6-A經由Sir2-dependent路徑延長酵母菌RLS 22
3.14 PN6-A經由TOR-independent路徑延長酵母菌RLS 23
3.15 PN6-A無法提高Sir2蛋白質表現量 23
3.16 PN6-A無法提升Pnc1蛋白質表現量 23
3.17 PN6-A提高SIRT1活性 24
3.18 PN6-A無法提高酵母菌NAD+濃度 24
第四章 結果與討論 25
圖表 28
參考文獻 60



圖目錄
附圖一. Damage accumulation in yeast ageing. 28
Fig. 1 Plumula Nelumbinis extract increases the viability of MEP strains in MEP analysis. 31
Fig. 2 Various concentrations of Plumula Nelumbinis extract increases the viability of MEP strains in MEP analysis. 32
Fig. 3 Various concentrations of Plumula Nelumbinis extract on replicative life span in Saccharomyces cerevisiae. 33
Fig. 4 Plumula Nelumbinis extract extends replicative life span via Sir2-dependent 34
pathway in Saccharomyces cerevisiae. 34
Fig. 5 Plumula Nelumbinis extract extends replicative life span via TOR-independent pathway in Saccharomyces cerevisiae. 35
Fig. 6 The Sir2 protein levels didn’t change following the treatment of Plumula Nelumbinis extract. 36
Fig. 7 The Pnc1 protein levels didn’t change following the treatment of Plumula Nelumbinis extract. 37
Fig. 8 Plumula Nelumbinis extract increases the activity of SIRT1. 38
Fig. 9 Plumula Nelumbinis extract augments the concentration of NAD+. 39
Fig. 10 Plumula Nelumbinis ethyl acetate layer increases the viability of MEP strains in MEP analysis. 40
Fig. 11 Compounds from the ethyl acetate layer of Plumula Nelumbinis. 43
Fig. 12 MEP assay of the pure compounds from Plumula Nelumbinis ethyl acetate extract. 47
Fig. 13 PN6 extends replicative life span in Saccharomyces cerevisiae. 48
Fig. 14 PN6 extends replicative life span via Sir2-dependent pathway in Saccharomyces cerevisiae. 49
Fig. 15 PN6 extends replicative life span via TOR-independent pathway in Saccharomyces cerevisiae. 50
Fig. 16 PN6-A extends replicative life span in Saccharomyces cerevisiae. 51
Fig. 17 Various concentrations of PN6-A on replicative life span in Saccharomyces cerevisiae. 52
Fig. 18 PN6-A extends replicative life span via Sir2-dependent pathway in Saccharomyces cerevisiae. 53
Fig. 19 PN6-A extends replicative life span via TOR-independent pathway in Saccharomyces cerevisiae. 54
Fig. 20 The Sir2 protein levels didn’t change following the treatment of PN6-A. 55
Fig. 21 The Pnc1 protein levels didn’t change following the treatment of PN6-A. 56
Fig. 22 PN6-A increases the activity of SIRT1. 57
Fig. 23 PN6-A can not augment the concentration of NAD+. 58
Fig. 24 Pathways by Plumula Nelumbinis extract and PN6-A extend the life span of yeast. 59
 
表目錄
Table 1 抗體資料表 11
Table 2 酵母菌株基因型資料表 11
Table 3. List of compounds isolated from Plumula Nelumbinis 29
Table 4. The structure of PN6 and PN6-A 30

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