臺灣博碩士論文加值系統

細胞自噬(autophagy)會影響老化的速率，在眾多長壽模式生物中，皆可發現細胞自噬能力有上升的現象，且被認為是延長壽命所需的重要因素之一，然而其中的分子調控機制還尚未完全明瞭。
飲食限制為延緩老化最有效的方式之一，過去的研究亦證實，其延長壽命的現象與細胞自噬的參與有關。其中，sams-1(S-adenosyl methionine synthetase-1)為飲食限制調控老化的重要基因，於本研究中，利用sams-1基因剔除的長壽線蟲作為實驗對象，經由觀察帶有螢光的細胞自噬標的蛋白LGG-1，在失去SAMS-1的情形下，會增加細胞自噬的活性。此外，當抑制細胞自噬形成的重要基因時，會縮短sams-1線蟲突變株延長的壽命，由此證實細胞自噬為SAMS-1調節壽命所需的要素。
HLH-30為人類TFEB(transcription factor EB)於線蟲中的同源基因，並被視為調控細胞自噬的重要轉錄因子以及影響老化的關鍵角色。在我們的研究中發現，利用RNAi抑制線蟲中的SAMS-1表現，會大幅提升HLH-30蛋白質表現及促進其進核的比率，而這樣的現象在餵食SAM(S-adenosyl methionine)之後則會明顯降低。同時我們也發現sams-1剔除的線蟲中壽命延長是需要HLH-30這個轉錄因子的作用。我們更進一步證實，HLH-30基因的表現量上升，是由於SAM缺乏引起SET-2活性下降，導致組蛋白甲基化改變所造成的。

Autophagy is an important cytoplasmic recycle process that removes damaged cellular components. It has been suggested that autophagy might be a common downstream effector in lifespan regulation. Indeed, studies have indicated that autophagy is required in dietary restriction (DR)-induced longevity in C. elegans. Previously, we have demonstrated that sams-1, S-adenosyl methionine synthetase-1, is a key regulator in lifespan extension induced by DR. Therefore, we asked whether autophagy might serve as one of the downstream effectors of SAMS-1 to mediate lifespan extension in C. elegans. In this study, we have found that RNAi knockdown or knockout of SAMS-1 significantly activates autophagy. We also observed that mRNA expression levels of autophagy-related genes are increased in sams-1 mutants. Moreover, RNAi knockdowns of autophagy genes, such as bec-1 and vps-34, decrease the lifespan of long-lived sams-1 mutants. Taken together, our findings suggest that elevating autophagy activity might be required for sams-1-mediated lifespan extension and that SAMS-1 might modulate autophagy through transcriptional regulation.
The TFEB orthologue, HLH-30, has shown to be an important transcription factor in regulating multiple autophagy-related genes in C. elegans. We found that RNAi knockdown of sams-1 largely increases the expression level and nucleus localization rate of HLH-30. However, nucleus localization rate of HLH-30 was diminished after extra supplementing of SAM(S-adenosyl methionine) in sams-1 RNAi mutants. We also demonstrate that HLH-30 is essential for the long lifespan of sams-1 mutants. Furthermore, our data indicate that SET-2 might regulate the transcriptional activity of HLH-30 through histone methylation.

目錄 Table of Contents

摘要 I
Abstract II
目錄 Table of Contents III
圖目錄 List of Figures IV
表目錄 List of Tables V
緒論 Introduction 1
研究目標 Aim 4
研究方法與材料 Methods and Materials 5
1. 線蟲(C. elegans)品系及培養 5
2. 線蟲壽命追蹤實驗 Lifespan Assay 6
3. GFP::LGG-1螢光點定量 Quantification of Puncta 8
4. 西方點墨法 Western Blot 8
5. 定量即時PCR Quantitative Real-Time PCR 9
6. 染色質免疫沉澱 Chromatin Immunoprecipitation 10
結果 Results 13
1. SAMS-1所調控的壽命延長現象需要細胞自噬的參與 13
2. HLH-30為SAMS-1影響老化的下游轉錄因子 14
3. SET-2藉由組蛋白的甲基化影響hlh-30的表現 15
討論 Discussion 18
圖表 Figures and Tables 20
參考文獻 References 52

圖目錄 List of Figures

圖 1. WT線蟲中表現GFP::LGG-1的螢光顯微鏡圖 20
圖 2. sams-1突變線蟲株中表現GFP::LGG-1的螢光顯微鏡圖 21
圖 3. sams-1突變線蟲株中表現GFP::LGG-1，經bec-1 RNAi處理後的螢光顯 微鏡圖 22
圖 4. 失去sams-1會促進細胞自噬的表現 23
圖 5. mCherry::GFP::LGG-1線蟲突變株的螢光顯微鏡圖 24
圖 6. sams-1 RNAi會增進autophagolysosome的表現量 25
圖 7. 抑制bec-1基因表現會縮短eat-2線蟲的壽命 26
圖 8. 抑制bec-1基因表現會縮短sams-1線蟲的壽命 27
圖 9. 抑制vps-34基因表現會縮短eat-2線蟲的壽命 28
圖 10. 抑制vps-34基因表現會縮短sams-1線蟲的壽命 29
圖 11. HLH-30::GFP線蟲突變株經sams-1 RNAi處理過後的螢光顯微鏡圖 30
圖 12. 抑制SAMS-1表現會導致HLH-30進核 31
圖 13. HLH-30::GFP線蟲突變株經sams-1 RNAi處理後，再餵食SAM的螢光顯微鏡圖 32
圖 14. SAM會影響HLH-30的進出核 33
圖 15. 抑制hlh-30基因表現會縮短sams-1線蟲的壽命 34
圖 16. SAMS-1減少會增加HLH-30的表現量 35
圖 17. 失去SAMS-1會促使hlh-30及其下游基因的mRNA表現量上升 36
圖 18. RNAi篩選結果量化圖 37
圖 19. GFP::LGG-1線蟲突變株餵食set-2 RNAi後的螢光顯微鏡圖 38
圖 20. set-2 RNAi會增進autophagosome的表現量 39
圖 21. SET-2減少會增加HLH-30的表現量 40
圖 22. 失去SET-2會促使hlh-30及lgg-1的mRNA表現量上升 41
圖 23. hlh-30基因序列位置圖 42
圖 24. 失去SET-2及SAMS-1會導致hlh-30啟動子上H3K4me3的甲基化減少 43
圖 25. sams-1及set-2線蟲突變株中W02C12.4片段的表現量呈現上升趨勢 44
圖 26. sams-1及set-2線蟲突變株中長鏈hlh-30 isoform的表現量呈現上升趨勢 45
圖 27. 抑制set-2基因表現並不會更加延長eat-2及sams-1線蟲的壽命 46
圖 28. 抑制hlh-30基因表現會縮短set-2線蟲的壽命 47

表目錄 List of Tables

表 1. 壽命追蹤分析及統計結果總表 48
表 2. HLH-30::GFP突變線蟲株經sams-1 RNAi處理後的進核比率表 50
表 3. HLH-30::GFP突變線蟲株經sams-1 RNAi處理後，再餵食SAM的進核比率表 51

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