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研究生:楊乃成
研究生(外文):Nae-Cherng Yang
論文名稱:細胞老化與能量限制:SA-βG方法的改良、老化模式的建立和菸鹼酸所扮演的角色
論文名稱(外文):Cell aging and caloric restriction: improvement of SA-βG assay, establishment of an aging model, and the role of niacin
指導教授:胡淼琳胡淼琳引用關係
指導教授(外文):Miao-Lin Hu
學位類別:博士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:90
中文關鍵詞:細胞老化基因消音理論菸鹼酸能量限制
外文關鍵詞:cell aginggene silencing theoryniacincaloric restriction
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根據許多酵母菌的研究,近來學者提出所謂的基因消音理論(gene silencing theory),因為這個理論可以解釋為何能量限制會延長壽命,因此引人注目。 但是,文獻上關於這個老化理論有兩種調控機制正在爭辯中,也就是NAD+ 波動模式(NAD+-fluctuation model)和nicotinamide (NAM)耗盡模式(NAM-depletion model)。雖然目前愈來愈多的證據證明NAM耗盡的模式在酵母中扮演主要的角色。然而,高等哺乳動物及人類細胞中是否存在同樣的調控機制,目前並不清楚。為了回答上述的問題,我們以人類的纖維母細胞(Hs68 cells)建立一個老化的細胞模式,並以2-deoxyglucose (一種能量限制模枋劑)和DHEA(一種被標榜具抗老化效果的營養補充劑雖然缺乏證據)的抗細胞老化作用之比較來驗證此細胞模式的可行性。我們也研究以senescence associated β-galactosidase (SA-βG) assay作為細胞老化標記的特異性,並研發出以fluorescein di-β-D-galactopyranoside (FDG)作為SA-βG受質的定量方法。然後我們進一步利用Hs68細胞模式來探討NAM和NA對細胞老化的影響,以及Hs68細胞培養在含不同濃度葡萄糖時(低糖可視為能量限制),NAD+含量或NAD+/NADH比例的變化。結果顯示NAM可延緩人類纖維母細胞Hs68的老化,而且在能量限制時,細胞中的NAD+或NAD+/NADH比例會增加。這些結果和文獻中以酵母菌為模式的研究報導有相當的出入,因為NAM在酵母中,而且在能量限制時,酵母的NAD+或NAD+/NADH比例維持不變。 這些結果也說明NAM耗盡的模式不適用於Hs68細胞中。 因此,雖然有些問題仍待進一步的釐清,我們的結果似乎是支持NAD+ 波動模式。本研究以Hs68細胞所獲得的結論不同於以酵母菌所得的結論說明Hs68細胞的老化機制與酵母菌的老化機制有所不同。
According to many yeast-based studies, a gene silence theory was proposed recently. The theory is interesting because it can explain how caloric restriction (CR) can extend lifespan. However, two different regulation mechanisms, i.e., the NAD+-fluctuation model and the nicotinamide (NAM)-depletion model for the aging theory are debated in the literature. To date, more and more evidence has shown that the NAM-depletion mechanism is favored in the yeast. However, it remains to be answered as to whether mammalian cells also possess the same mechanisms as does the yeast. For revealing the question, we established a cell-aging model using human foreskin fibroblast Hs68 cells. The availability of model was validated by comparing anti cell-aging ability of two agents of 2-deoxyglucose, a caloric restriction mimic, and DHEA, a nutritional supplement for anti-aging commercially but without sufficient evidence. We also investigated the specificity of the senescence associated β-galactosidase (SA-βG) assay as a marker for cell aging and developed a new quantitative method for determining SA-βG activity using fluorescein di-β-D-galactopyranoside (FDG) as a substrate. Using the aging-cell model, we further studied the effects of NAM and nicotinic acid (NA) on the aging of Hs68 cells, and the NAD+ level or NAD+/NADH ratio fluctuation corresponding to the cells cultured in the mediums with different glucose concentrations (the low glucose medium can be considered as caloric restriction). The results showed NAM could retard senescence of human foreskin fibroblast Hs68 cells. In addition, the cellular NAD+ levels as well as NAD+/NADH ratios increased in the glucose (calorie)-restricted cells. These results were different from that of yeast studies, as reported in the literature, because the NAD+ levels or NAD+/NADH ratios would not change in the glucose-restricted yeast. The results illustrated that the NAM-depletion model could not be applied to Hs68 cells and it appeared to support the NAD+-fluctuation model but also raised several questions regarding the interpretation of the model. In conclusion, our results on Hs68 cell aging are different from those on yeast as reported in the literature, suggesting that the aging mechanims of the two cells systems may be different.
Contents
Abbreviations 1
中文摘要 2
Overall abstract 3
Overall introduction 4
Chapter I
A fluorimetric method using fluorescein di-β-D-galactopyranoside for quantifying the senescence associated-β-galactosidase activity in human foreskin fibroblast Hs68 cells. [Anal. Biochem. (2004) 325: 337-343] 11
Chapter II
The limitations and validities of senescence associated-β-galactosidase activity as an aging marker for human foreskin fibroblast Hs68 cells 28
Supplement 1 43
Supplement 2 44
Chapter III
2-Deoxyglucose, a caloric restriction mimic, but not dehydroepiandrosterone, retards senescence of human foreskin fibroblast Hs68 cells: establishment of a cell-aging model 45
Supplement 3 63
Supplement 4 64
Chapter IV
Caloric restriction, niacin and NAD+ in aging of human foreskin fibroblast Hs68 cells 65
Overall conclusion 83
Appendix I
1. DHEA inhibits cell growth and induces apoptosis in BV-2 cells and the effects are inversely associated with glucose concentration in the medium. [J. Steroid Biochem. Mol. Biol. (2000) 75: 159-166] 84
2. ATP depletion is an important factor in DHEA-induced growth inhibition and apoptosis in BV-2 cells. [Life Science (2002) 70: 1979-1988]. 85
3. A convenient one-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP. [Anal. Biochem. (2002) 306: 323-327] 86
Supplement 5 87
Appendix II
1) Resume of Nae-Cherng Yang 88
2) Publication list of Nae-Cherng Yang (~2004) 89
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