臺灣博碩士論文加值系統

根據台灣行政院內政部估計，台灣大於65歲的老年人口比例將從2010年的10.7%大幅提升至2030年的24.4%。也就代表著在未來的20年，台灣可能會有大於目前兩倍的老年人可能受到老年相關疾病所苦。因此對於如何減緩老化過程以及老化相關疾病的議題也持續受到科學家的關注及興趣。除此之外，研究也發現，老化過程所牽涉的分子路徑與老化相關疾病的致病機轉路徑極為相似，因此可用於延長壽命的藥物或是食療有極大的可能性也可用於治療老化相關的疾病。老化相關模組中酵母菌的老化定義清楚，為廣泛運用模組之一，本實驗室先前發現補骨脂具有延長酵母菌壽命的功效，因此本論文利用mother enrichment program (MEP)快篩系統，篩選出corylin可能為有效成分之一。接著以RLS (replicative life span) analysis驗證corylin是否可增加酵母菌的RLS。結果也顯示，corylin具有延長酵母菌RLS效果，並進一步發現corylin利用Sir2-dependent及Sir2-independent路徑延長酵母菌的RLS。而在給予corylin後，酵母菌Pnc1的表現上升與限制卡路里(Calorie Restriction, CR)表現相似。我們也發現，將酵母菌進行CR並給予corylin時，酵母菌RLS並無疊加效果，顯示corylin增加酵母菌RLS與calorie restriction的路徑相似。除此之外，corylin也可以在人類癌症細胞株A549及U2OS中刺激SIRT1的活性，顯示corylin可能延緩人類細胞衰老。綜合以上結果，corylin在延長壽命及治療老化相關疾病上具有相當大的潛力。

The ROC Minister of the Interior estimated that the percentage of people aged 65 years and older in Taiwan will increased from 10.7% in 2010 to 24.4% in 2030. The number of people suffering from aged-related diseases is anticipated to more than double over the next two decades. Interventions that can decelerate aging processes continue to raise interest among medical research. Besides, the molecular mechanisms underlying aging appear to be interconnected the same pathways with age-related diseases. Therefore, drugs and dietary regimens that can extend lifespan could potentially be used to treat age-related diseases. The budding yeast Saccharomyces cerevisiae has been used extensively to study the fundamental processes of aging and age-related study. Our previous work found that ethanol extract of Psoralea corylifolia extends replicative life span in yeast. By using a high throughput system, mother enrichment program (MEP), we first identified an active compound called corylin that isolated from Psoralea corylifolia. Next we confirmed the result of MEP and found that corylin increased life span in yeast via Sir2-dependent and Sir2-independent pathway by using replicative life span analysis. Following corylin treatment, Pnc1 expression was increased that similar to calorie restriction (0.5% glucoses). Furthermore, corylin failed to increase replicative life span in calorie restriction condition suggesting that corylin may increase replicative life span by mimicking calorie restriction in yeast. Moreover, we showed that corylin stimulated SIRT1 activity in A549 and U2OS cell lines. Moreover, corylin decreased cellular senescence in human umbilical vein endothelial cells indicating that corylin may extend lifespan in mammalian cells. Therefore, corylin could potentially be used to extend lifespan in human and to treat age-related diseases.

目錄
指導教授推薦書
口試委員審定書
致謝 iii
中文摘要 iv
Abstract v
目錄 i
圖目錄 iv
表目錄 vi
縮寫表 vii
研究動機 1
第一章簡介 2
1.1前言 2
1.2酵母菌於老化研究上優勢 2
1.3 Chronological life span (CLS)及Replicative life span (RLS) 2
1.4 Mother enrichment program (MEP) 3
1.5 Extrachromosomal ribosomal DNA circles (ERCs) 3
1.6卡路里限制 (Calorie restriction；CR) 4
1.7 Silent Information Regulator 2 (Sir2) 5
1.8 Pnc1 5
1.9 Sirtuin1 (SIRT1) 5
1.10 p53 6
1.11補骨脂 6
第二章實驗材料與方法 14
2.1實驗材料 14
2.1.1藥品 14
2.1.2酵母菌株 14
2.1.3酵母菌基因型資料表 14
2.2實驗方法 15
2.2.1 酵母菌培養 15
2.2.2 細胞培養 16
2.2.3 Yeast transformation 16
2.2.4 Liquid aging assay 17
2.2.5 Replicative life span analysis 17
2.2.6 Whole cell protein extracts 18
2.2.7 Yeast TCA whole cell extracts 18
2.2.8 Western blot 18
2.2.9 SIRT1 in vitro activity assay 19
2.2.10 RNA extracts 19
2.2.11 Reverse transcription polymerase chain reaction (RT-PCR) 20
2.2.12 Immunofluorescence microscopy 20
2.2.13 SA-β-GAL染色 20
第三章結果 21
3.1利用MEP系統篩選補骨脂的活性成分 21
3.2 Corylin增加酵母菌的RLS 22
3.3 Corylin經由Sir2-dependent及Sir2-independent路徑延長酵母菌RLS 23
3.4 Corylin間接活化SIRT1 24
3.5 Corylin提升Sir2上游Pnc1的蛋白質表現量 24
3.6 Corylin具濃度依賴性提升Pnc1蛋白質表現量 25
3.7 Corylin經由mimic CR增加酵母菌RLS 25
3.8 Corylin在人類細胞株中活化SIRT1 26
3.9 Corylin 延緩人類細胞衰老 27
第四章結果與討論 29
參考文獻 52

圖目錄
附圖 1. Psoralea corylifolia (FP)extends the replicative life span in yeast. 7
附圖 2. The column chromatography of Psoralea corylifolia hexane layer extract. Extraction flow chart of Psoralea corylifolia. 8
附圖 4. Damage accumulation in yeast ageing. 12
附圖5. Viability curves of haploid yeast strains generated by micromanipulation (A) and liquid culture with the MEP (B). 13
Fig. 1 MEP assay of the fractions from Psoralea corylifolia hexane extract. 36
Fig. 2 Corylin extends replicative life span in Saccharomyces cerevisiae. 37
Fig. 3 Various concentrations of corylin on replicative life span in Saccharomyces cerevisiae. 38
Fig. 4 Corylin extends replicative life span via Sir2-dependent and Sir2-independent pathway in Saccharomyces cerevisiae. 40
Fig. 5 Corylin does not activated SIRT1 in vitro. 41
Fig. 6 Corylin up regulates the Pnc1 protein level in time dependent manner. 42
Fig. 7 The transcriptional regulation of Pnc1 is affected by corylin in Time-dependent manner. 43
Fig. 8 Corylin up regulates the Pnc1 protein level in dose dependent manner. 44
Fig. 9 Corylin extends lifespan by mimicking Calorie Restriction (CR). 46
Fig. 10 Corylin inactivates UV-induced acetylation of p53 in A549 cell. 47
Fig. 11 Corylin inactivates UV-induced acetylation of p53 in U2OS cell. 48
Fig. 12 Corylin increases the population doubling level (PDL) of human umbilical vein endothelial cell (HUVECs). 49
Fig. 13 Senescence-associated-β-galactosidase (SA-β-gal)-positive HUVECs were significantly decreased following corylin treatment. 50
Fig. 14 Inhibition of p21, phospho-mTOR and phospho-S6K by corylin. 51

表目錄
Table 2.1 酵母菌株基因型資料表 14
Table 2.2 引子序列資料表 15
 

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