臺灣博碩士論文加值系統

動物對抗環境及生理壓力的能力與能否長壽有很大的關係，The Insulin/IGF-1-like signaling（IIS）pathway是目前已知動物對抗壓力和延長壽命相關的重要機制。線蟲中IIS訊息傳遞途徑可以負調控DAF-16/FOXO轉錄因子的活性，當IIS的訊息變弱時，會引發DAF-16/FOXO活化入核並延長壽命。
IIS另一個重要的下游因子是熱休克轉錄因子HSF-1， HSF-1可以調控壽命以及參與熱休克反應。先前研究已知HSF-1過度表現時會延長線蟲壽命，而當RNAi knockdown時則壽命縮短，且和DAF-16一樣，在IIS途徑中是需要HSF-1的活化才能促使壽命延長。先前研究指出，只需在神經中過度表現HSF-1就足以達到壽命延長，而過程中需要活化腸道中的DAF-16，代表著神經細胞中的HSF-1可能以非細胞自我調控（Cell-nonautonomous Regulation）的方式影響腸道中的DAF-16。而我們實驗室也證實當在神經過度表現HSF-1時，的確會造成腸道的DAF-16進核並且活性上升。我們接下來希望能進一步了解這個現象是需要所有的神經參與，或是只需要特定的神經細胞中的HSF-1活化即可造成腸道DAF-16進核，進而使線蟲壽命延長。
為了尋找哪一類神經HSF-1參與調控腸道DAF-16，我們建構出特定神經過度表現HSF-1的線蟲，觀察是否影響線蟲壽命與腸道DAF-16的進出核。我們發現，神經HSF-1 RNAi knockdown時，不會影響線蟲壽命。然而，當Cholinergic神經過度表現HSF-1時，足以延長線蟲壽命，但不會造成腸道DAF-16的進核。此外，需要分泌神經傳導物質與神經肽的神經HSF-1共同參與下，才能造成腸道DAF-16的進核。因此，調控腸道DAF-16的進核與壽命的機制為兩條獨立的路徑。未來會進一步探討調控腸道DAF-16的神經是哪些並且是如何溝通。

Tissue-tissue communications are integral to organismal aging, orchestrating a body-wide aging process. The insulin/IGF-1-like signaling pathway (IIS) is known to have a conserved role in aging. IIS negatively regulates the activity of the FOXO transcription factor DAF-16 and the expression of its downstream target genes. In particular, DAF-16/FOXO activity in the intestinal cells is required for the lifespan extension of the IIS pathway mutants.
Heat shock factor 1 (HSF1) is an evolutionarily highly conserved transcription factor that could prevent cellular degeneration and increase thermal tolerance by regulating the expression of the heat shock responsive genes. HSF-1 is also a master regulator in aging process. It is known that HSF-1 and DAF-16/FOXO work together to enhance lifespan extension in response to reduced DAF-2 insulin-like signaling. Previous studies have also indicated that neural hsf-1 functions cell non-autonomously to regulate longevity via activation of intestinal daf-16. However, the molecular mechanisms by which neuronal HSF-1 modulates intestinal DAF-16 are mostly unclear.
Here, we further identified the key neurons that mediate the crosstalk between neural HSF-1 and intestinal DAF-16 in the regulation of longevity and stress responses. First, we confirmed that nuclear translocation of intestinal DAF-16 is activated in response to neural HSF-1 overexpression, and eventually extends lifespan and increases the ability of stress resistance. Moreover, we found that knockdown hsf-1 in neurons does not shorten the longevity phenotypes. However, cholinergic neural overexpression of HSF-1 is sufficient to promote longevity and also requires the activities of DAF-16, whereas it does not trigger nuclear translocation of intestinal DAF-16. In addition, it needs more than two kinds of neurons overexpressing HSF-1 to activate the nuclear translocation of intestinal DAF-16. Thus, nuclear translocation of intestinal DAF-16 and age regulation are separate pathways. Further investigation is needed to research which neurons communicate with DAF-16 in the intestine.

目 錄
摘要……………………………………………………………….......................……….i
ABSTRACT……...……………………………………………………........................ii
目錄…………………………………………………………………….........................iv
圖目錄………………………………………………………………......................…….v
表目錄………………………………………………………………......................…...ix
縮寫列表…………………………………………………………......................………x
緒論………………………………………………………….....................….…………..1
材料方法……………………………………………………......................……………4
實驗結果………………………………………………………….....................……..13
討論……………………………………………………………….....................……...27
圖表…………………………………………………………….....................………...32
附表……………………………………………………….....................……………...94
參考文獻…………………………………………………………….....................…..99
附錄…………………………………………………………...................…………...103

圖 目 錄
Figure 1-1. rgef-1p::hsf-1::sl2::mCherry ; daf-16::gfp 突變株的螢光
照相結果......................................................................32
Figure 1-2. 神經過度表現HSF-1會促使腸道DAF-16進核…….....…….33
Figure 1-3. 神經過度表現HSF-1會活化DAF-16的活性……………..…..34
Figure 1-4. 神經過度表現HSF-1足以延長線蟲壽命…………….…….…..35
Figure 1-5. 神經過度表現HSF-1在基因插入特定染色體處理
（Integration）後仍可以延長線蟲壽命…..…………....……..36
Figure 1-6. 神經過度表現HSF-1增加線蟲的壓力抵抗能力………...…..37
Figure 2-1. unc-25p::hsf-1::sl2::YFP; daf-16::gfp 突變株表現的神經
細胞分佈.....................................................................38
Figure 2-2. unc-25p::hsf-1::sl2::YFP; daf-16::gfp 突變株的螢光照相
結果……......................................................................39
Figure 2-3. GABAergic神經過度表現HSF-1不會造成腸道DAF-16進
核...............................................................................40
Figure 2-4. unc-25p::hsf-1::sl2::YFP; daf-16::gfp突變株的腸道DAF-
16核質比定量結果……………………………..............…….......41
Figure 2-5. GABAergic神經過度表現HSF-1不會影響線蟲壽命….…..42
Figure 3-1. eat-4p::hsf-1::sl2::mCherry; daf-16::gfp 突變株表現的神
經細胞分佈…...…………………………………..........………….….43
Figure 3-2. eat-4p::hsf-1::sl2::mCherry; daf-16::gfp 突變株的螢光
照相結果.....................................................................44
Figure 3-3. Glutamatergic神經過度表現HSF-1不會造成腸道DAF-16
進核….....................................................................….45
Figure 3-4. eat-4p::hsf-1::sl2::mCherry; daf-16::gfp突變株的腸道
DAF-16核質比定量結果…………………………….....…………..46
Figure 3-5. Glutamatergic神經過度表現HSF-1不會影響線蟲壽命...47
Figure 4-1. tph-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株表現的神
經細胞分佈……………..………………………………........…...…...48
Figure 4-2. tph-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株的螢光
照相結果…...................................................................49
Figure 4-3. Serotonergic神經過度表現HSF-1不會造成腸道DAF-16進
核…….......................................................................…50
Figure 4-4. tph-1p::hsf-1::sl2::mCherry; daf-16::gfp突變株的腸道
DAF-16核質比定量結果………………………………….…...……51
Figure 4-5. Serotonergic神經過度表現HSF-1不會影響線蟲壽命.....52
Figure 5-1. tdc-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株表現的神
經細胞分佈…………………………….....……..........………………53
Figure 5-2. tdc-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株的螢光照
相結果.........................................................................54
Figure 5-3. Tyraminergic/ Octopaminergic神經過度表現HSF-1不會
造成腸道DAF-16進核………………………………………….......55
Figure 5-4. tdc-1p::hsf-1::sl2::mCherry; daf-16::gfp突變株的腸道
DAF-16核質比定量結果…………………………...…........…….56
Figure 5-5. Tyraminergic/ Octopaminergic神經過度表現HSF-1不會
影響線蟲壽命……….............……….....…………………………..57
Figure 6-1. dat-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株表現的神
經細胞分佈……………………………………................……...….58
Figure 6-2. dat-1p::hsf-1::sl2::mCherry; daf-16::gfp 突變株的螢光照
相結果….....................................................................59
Figure 6-3. Dopaminergic神經過度表現HSF-1不會造成腸道DAF-16
進核…......................................................................…60
Figure 6-4. dat-1p::hsf-1::sl2::mCherry; daf-16::gfp突變株的腸道
DAF-16核質比定量結果……………………....…………………..61
Figure 6-5. Dopaminergic神經過度表現HSF-1不會延長線蟲壽命….62
Figure 7-1. unc-17p::hsf-1::sl2::mCherry; daf-16::gfp 突變株表現的
神經細胞分佈……………………………………………….....………..63
Figure 7-2. Cholinergic神經過度表現HSF-1不會造成腸道DAF-16進
核……..........................................................................64
Figure 7-3. unc-17p::hsf-1::sl2::mCherry; daf-16::gfp突變株的腸道
DAF-16核質比定量結果……………………………………………..65
Figure 7-4. Cholinergic神經過度表現HSF-1不會影響線蟲的壓力抵抗
能力….........................................................................66
Figure 7-5. Cholinergic神經過度表現HSF-1會延長線蟲壽命….....….67
Figure 7-6. acr-2p::hsf-1::sl2::CFP; daf-16::gfp 突變株表現的神經細
胞分佈…......................................................................68
Figure 7-7. acr-2p::hsf-1::sl2::CFP; daf-16::gfp 突變株能有效表現
CFP的基因片段…….........………………………………………..…69
Figure 7-8. acr-2p::hsf-1::sl2::CFP; daf-16::gfp 突變株的螢光照相結
果…….......................................................................….70
Figure 7-9. Cholinergic神經過度表現HSF-1（acr-2p::hsf-1 O.E.）不
會造成腸道DAF-16進核………………………………………..……71
Figure 7-10. acr-2p::hsf-1::sl2::CFP; daf-16::gfp突變株的腸道DAF-
16核質比定量結果……………………………………………….….72
Figure 7-11. Cholinergic神經過度表現HSF-1（acr-2p::hsf-1 O.E.）足
以延長線蟲壽命..……………………………………….…………....73
Figure 8. 神經過度表現HSF-1需要DAF-16來延長線蟲壽命….…………74
Figure 9. Cholinergic神經過度表現HSF-1足以延長線蟲壽命並且需要
DAF-16的活化…………………………………………………………....75
Figure 10-1. unc-17p::hsf-1 O.E.; eat-4p::hsf-1 O.E.; tph-1p::hsf-1
O.E.; tdc-1p::hsf-1 O.E.; dat-1p::hsf-1 O.E.; unc-
25p::hsf-1 O.E.; daf-16::gfp 突變株表現的神經細胞分
佈……………........................………………………………………76
Figure 10-2. 分泌神經傳導物質的神經中過度表現HSF-1會造成腸道
DAF-16明顯進核…..…………………………………………………77
Figure 11-1. 神經增強hsf-1 RNAi knockdown會縮短線蟲壽命……....78
Figure 11-2. hsf-1 RNAi能有效降低hsf-1 mRNA的表現量…………..…79
Figure 12. GABAergic神經hsf-1 knockdown不會影響線蟲壽命…....80
Figure 13. Glutamatergic神經hsf-1 knockdown不會影響線蟲壽命..81
Figure 14. Dopaminergic神經hsf-1 knockdown不會影響線蟲壽命..82
Figure 15. Cholinergic神經hsf-1 knockdown不會影響線蟲壽命…...83
Figure 16-1. Serotonergic 神經RNAi knockdown突變株表現的神經細
胞分佈.......................................................................84
Figure 16-2. Serotonergic神經hsf-1 knockdown不會影響線蟲壽
命..............................................................................85
Figure 17-1. Tyraminergic/ Octopaminergic 神經RNAi knockdown突
變株表現的神經細胞分佈………………..............…………....86
Figure 17-2. Tyraminergic/ Octopaminergic神經hsf-1 knockdown不
會影響線蟲壽命………….....……………………………………....87
Figure 18-1. 神經RNAi knockdown 突變株給予rab-3 RNAi後的照相結
果…...........................................................................88
Figure 18-2. 神經RNAi knockdown突變株具有RNAi knockdown的效
果……........................................................................89
Figure 18-3. 神經hsf-1 RNAi knockdown不會影響線蟲壽命…………90
Figure 19. 調控腸道DAF-16進核與長壽的機制為兩條獨立的路徑.…..91
Figure 20. 神經增強RNAi knockdown突變株會影響肌肉組織的功
能.................................................................................92
Figure 21. 腸道hsf-1 RNAi knockdown會縮短線蟲壽命…………...…..93

表 目 錄
Table 1. 神經細胞分類與相對應的特定神經啟動子………….…..…........94
Table 2. 過度表現HSF-1於氧化壓力及壽命分析的實驗記錄……........95
Table 3. HSF-1、DAF-16 Knockdown於壽命分析的實驗記錄…....…97

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