臺灣博碩士論文加值系統

在市售丙酮酸膳食補充品中的不純物 parapyruvate，具有抑制α-ketoglutarate dehydrogenase complex (KGDHC)活性的作用。研究發現 KGDHC活性的降低是發生神經退化性疾病重要的機制之一，我們先前研究發現，添加 parapyruvate可抑制KGDHC明顯地縮短人類Hs68細胞的壽命，顯示KGDHC在細胞衰老扮演重要角色，然而，parapryvuate是否誘發線蟲(C. elegans)老化以及KGDHC是否在線蟲的老化扮演重要角色目前並不清楚。本研究以不同劑量的parapyruvate (0、3.5、35、106μg/plate)處理線蟲，結果發現高劑量的parapyruvate (106μg/plate)可以顯著的抑制線蟲體內的KGDHC活性，並且顯著的縮短線蟲壽命，而KGDHC的活化劑鈣離子則可以拮抗高劑量parapryvuate所誘發的縮短壽命的作用。此外，一種KGDHC線蟲的突變株Tm6589/nt1，其KGDHC活性較正常線蟲約減少40%，亦顯示顯著地縮短線蟲壽命的作用，綜合上述的結果顯示高劑量的parapyruvate可以藉由抑制KGDHC而引發線蟲衰老並且KGDHC在線蟲的老化扮演重要角色，然而，低劑量(3.5μg/plate)的parapyruvate可以輕微的抑制KGDHC，卻顯示延長線蟲的壽命。進一步，我們使用aak-2、sir-2.1、jnk-1、skn-1、daf-15及akt-1等突變株，探討低劑量延長壽命及高劑量parapyruvate縮短壽命所誘發的訊息傳遞機制，結果發現低劑量parapryuvate延長壽命現象，在aak-2、sir-2.1、jnk-1、daf-15突變株會消失，顯示低劑量parapryuvate延長壽命可能和模擬熱量限制有關；而高劑量parapyruvate縮短壽命的作用，在jnk-1和skn-1突變株會消失，甚至在jnk-1突變株高劑量的parapyruvate相反地會使壽命延長，顯示高劑量parapryuvate縮短線蟲的作用，可能與氧化壓力升高有關。

Parapyrurvate, an impurity in commercially available pyruvate dietary supplements, has an effect of inhibiting the activity of α-ketoglutarate dehydrogenase complex (KGDHC). Studies have found that the reduction of KGDHC activity is one of the important mechanisms of neurodegenerative diseases. Our previous studies found that the addition of parapyruvate inhibited KGDHC and significantly shortened the lifespan of human Hs68 cells, indicating that KGDHC played an important role in cellular senescence. However, it is unclear whether parapryvuate has ability to induce the aging of C. elegans and whether KGDHC plays an important role in the aging of C. elegans. In this study, C. elegans were treated with different doses of parapyruvate (0、3.5、 35、 106μg/plate), and it was found that the high dose of parapyruvate (106μg/plate) significantly inhibited KGDHC activity in C. elegans and significantly shortened the lifespan of C. elegans. In addition, the calcium ion, a KGDHC activator, could antagonize the lifespan-shortening effects induced by the high dose of parapryvuate. Moreover, Tm6589/nt1, a KGDHC mutant of C. elegans, had a KGDHC activity reduced by about 40% compared with N2, and it also showed a significant shortening in lifespan. The above results showed that the high dose of parapyruvate could induce the aging of C. elegans by inhibiting KGDHC activity and KGDHC played an important role in the aging of C. elegans. However, studies have found that the low dose (3.5 μg/plate) of parapyruvate could slightly inhibit KGDHC but significantly extended the lifespan of the C. elegans. Further, we used aak-2, sir-2.1, jnk-1, skn-1, daf-15 and akt-1 mutants to investigate the signaling mechanisms for both the lifesapn-prolonged effect of low-dose parapryuvate and the lifespan-shortening effect of high-dose parapyruvate. It was found that the lifespan-prolonged effect of low-dose parapryuvate was disappeared in aak-2, sir-2.1, jnk-1and daf-15 mutants, indicating that the lifespan-prolonged effect of low-dose parapryuvate may be related to the caloric restriction-mimicking effects. The lifespan-shortening effect of high-dose parapyruvate was also disappeared in jnk-1 and skn-1 mutants, and even the high dose of parapyruvate in turn prolonged the lifespan of jnk-1 mutant, indicating that the lifespan-shortening effect of high-dose parapyruvate may be associated with the increased oxidative stress.

謝誌 ii
中文摘要 I
Abstract III
目錄 V
表次 VIII
圖次 IX
縮寫檢索表 1
前言 2
文獻探討 4
1. 丙酮酸鹽膳食補充品(pyruvate dietary supplumnets) 4
2. Parapyruvate (2-hydroxy-2-methyl-4-oxopentanedioic acid) 4
3. α-酮戊二酸去氫酶複合體 (α-ketoglutarate dehydrogenase complex , KGDHC) 5
4. 胰島素/類胰島素生長因子-1信號傳導 (IIS pathway) 6
5. c-Jun N-末端激酶(The c-Jun N-terminal kinase；JNK pathway 7
6. 單磷酸腺苷活化蛋白質激酶( (AMP activated protein kinase ；AMPK pathway) 8
7. mTOR pathway 9
8. 活性氧化物質 (Reactive oxygen species, ROS) 9
9. Sirtuin 10
10. 秀麗隱桿線蟲 (Caenorhabditis elegans) 10
11. 研究動機與目的 11
一、實驗架構 13
二、材料與方法 14
1. 藥品試劑 14
2. 線蟲種類及來源 15
3. E. coli 培養與製備 15
4. 線蟲壽命模式 16
5. 線蟲KGDHC試驗 17
6. 蛋白質測定 18
7. 線蟲游泳試驗 (活動力試驗) 18
8. 線蟲咽喉跳動試驗 (活動力試驗) 19
9. 自體螢光試驗(Lipofuscin的紅光發射和NADH的藍光發射之測定) 19
10. 統計分析 19
三、結果 20
1. Parapyruvate對N2線蟲之壽命影響(trial 1) 20
2. Parapyruvate對N2線蟲之壽命影響(trail 2) 20
3. N2線蟲生理試驗 20
4. Parapyruvate體外及體內抑制N2線蟲KGDHC活性的能力 21
5. KGDHC突變株Tm6589/nt1生命期試驗 21
6. KGDHC突變株Tm6589/nt1生理試驗 22
7. KGDHC突變株體內KGDHC活性試驗 22
8. Ca2+拮抗高劑量parapyruvate延長蟲壽命的作用 22
9. Ca2+拮抗低劑量parapyruvate延長線蟲壽命的作用 23
10. Parapyruvate對aak-2突變株之壽命試驗 23
11. Parapyruvate對sir2.1突變株之壽命試驗 23
12. Parapyruvate對jnk-1突變株之壽命試驗 23
13. Parapyruvate對skn-1突變株之壽命試驗 24
14. Parapyruvate對daf-15突變株之壽命試驗 24
15. Parapyruvate對akt-1突變株之壽命試驗 24
四、討論 26
五、結論 29
六、參考文獻 30
七、實驗圖表 37

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