臺灣博碩士論文加值系統

能量限制(calorie restriction; CR)具有延長生物體壽命的作用。菸鹼醯胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide; NAD+)是一種活化態的維生素B3。 Sirtuins是一群NAD+-dependent deacetylase，其中SIRT1具有延長生物體壽命的能力。FK866是NAD+合成救援路徑中nicotinamide phosphoribosyltransferase (NAMPT)的抑制劑，可以降低細胞內NAD+濃度。本研究的目的在於釐清CR是否藉由增加細胞內的NAD+濃度，進而活化SIRT1活性，而能延長細胞壽命。利用人類Hs68細胞培養在含低濃度葡萄糖的培養基(glucose restriction; GR)來模擬CR，我們比較GR、NAD+及FK866對細胞的壽命及細胞內NAD+濃度的影響，並探討FK866對細胞的作用是否被GR及在培養基中添加NAD+所拮抗。細胞壽命(replicative lifespan)以累積性生長曲線來測定，senescence associated β-galactosidase (SA-βG)活性染色則作為細胞衰老的生物標記，細胞內NAD+的濃度以酸萃取結合酵素循環呈色法測定，SIRT1的活性以商業化的試劑組或以西方墨點法及免疫沉澱法偵測p53的乙醯化程度來測定。結果發現GR可以顯著的增加細胞內NAD+濃度，進而活化SIRT1並延長細胞的壽命。而添加NAD+亦可顯示與GR相似的作用。相反的，處理FK866則造成細胞內NAD+濃度顯著的下降，並且縮短細胞壽命及增加SA-βG活性。重要的是，FK866所誘發的NAD+降低、細胞壽命縮短及SIRT1活化等作用，皆可有效的被GR及添加NAD+所拮抗。此結果顯示細胞內NAD+濃度的增加，在CR延長生物體壽命機制中扮演重要角色。

Caloric restriction (CR) can extend the lifespan of organisms. Nicotinamide adenine dinucleotide (NAD+) is one of bioactive from of vitamin B3. Sirtuins is a protein family called NAD+-dependent deacetylase, which is considered as a longevity protein. FK866 is an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), which is the rate limiting enzyme in the salvage pathway of NAD+ synthesis from nicotinamide and has ability to decrease intracellular NAD+ level. In this study, we examined whether CR can increase intracellular NAD+ levels, and then activate SIRT1 and extend replicative lifespan of human Hs68 cells. Using cells cultured at lower levels of glucose than normal level of glucose in medium (glucose restriction; GR) to mimic CR, we compared the effects of GR, NAD+ and FK866 on cell lifespan, intracellular NAD+ levels, SIRT1 activity and senescence associated -galactosidase (SA-G) activity. We also examined whether the effects of FK866 on Hs68 cells can be antagonized by GR and adding NAD+ into the culture medium. The lifespan of cells was measured as the cumulated growth curve of population doubling levels. The biomarker of cell senescence was detected by determination of senescence associated -galactosidase (SA-G) activity using X-Gal staining. Intracellular NAD+ was determined by an acid extraction method and followed by an enzymatic cycling method. SIRT1 activities were determined by SIRT1 Direct Fluorescent Screening Assay Kit or used p53 acetylation to represent cellular activities of SIRT1. The results showed that GR (>5day) could significantly increase NAD+ levels in cells, activated SIRT1 activity, and extended the lifespan of Hs68 cells. Add NAD+ in to the medium showed with similar effects as GR. Oppositely, FK866 has abilities to reduce intracellular NAD+ levels, shorten the cells lifespan, decrease SIRT1 activity, and increase SA-βG activity. Importantly, all of these effects of FK866 on Hs68 cells can be effectively blocked by GR or adding NAD+ into the medium. The results demonstrated that increase of intracellular NAD+ level plays a crucial role in the mechanism of CR-extended lifespan of human Hs68 cells.

中文摘要........................................................................................................................ii
英文摘要.......................................................................................................................iii
目錄...............................................................................................................................v
縮寫檢索表....................................................................................................................1
圖表目錄........................................................................................................................3

一、前言........................................................................................................................5
二、文獻回顧................................................................................................................7
1. 能量限制...........................................................................................................7
2. SIRT1..................................................................................................................8
3. NAD+..................................................................................................................9
4. 細胞老化模式.................................................................................................12
三、實驗架構................................................................................................................13
四、材料與方法............................................................................................................14
1. 藥品試劑.........................................................................................................14
2. 細胞解凍及保存.............................................................................................15
3. 細胞毒性及細胞生長之檢測.........................................................................15
3.1 細胞存活率檢測.......................................................................................15
3.2 細胞生長檢測...........................................................................................15
4. 細胞內NAD+ 濃度測定................................................................................17
5. 細胞壽命(replicative lifespan)檢測................................................................18
6. 細胞老化相關的β-半乳糖苷酶(SA-G)活性檢測....................................18
7. 細胞內SIRT1活性測定................................................................................20
7.1 以SIRT1 kit測定活性..............................................................................20
7.2 以Western blotting測定細胞中acetyl p53代表SIRT1活性...................21
7.3 以免疫沉降法測定acetyl p53代表SIRT1活性......................................24
8. 統計分析.........................................................................................................24
五、結果........................................................................................................................25
1. GR、NAD+及FK866對細胞存活率之影響.................................................25
2. GR、NAD+及FK866對細胞生長之影響.....................................................25
3. GR及NAD+拮抗FK866所誘發的細胞生長抑制作用................................26
4. GR、NAD+及FK866對細胞內NAD+濃度的影響......................................26
5. GR及NAD+拮抗FK866降低細胞內NAD+濃度的作用.............................27
6. GR、NAD+及FK866對細胞壽命的影響及拮抗的作..................................27
7. GR及NAD+拮抗FK866誘發之細胞衰老形態及密度降低的情形............28
8. GR及NAD+拮抗FK866增加SA-G活性染色的情形............................28
9. GR、NAD+與FK866對SIRT1活性的影響及拮抗的作用
—以SIRT1活性套組測定.................................................................................29
10. GR及NAD+拮抗FK866對SIRT1活性的影響
—以西方墨點法測定acetyl p53/p53.................................................................29
11. GR及NAD+拮抗FK866對SIRT1活性的影響
—以免疫沉澱法測定acetyl p53/p53...................................................................30
12. GR、NAD+與FK866對SIRT1蛋白表現的影響..............................................30
六、討論........................................................................................................................31
七、結論.......................................................................................................................36
八、圖表.......................................................................................................................37
九、參考文獻...............................................................................................................52

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