跳到主要內容

臺灣博碩士論文加值系統

(35.175.191.36) 您好!臺灣時間:2021/08/02 13:14
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:余佳欣
研究生(外文):Jia-Sin Yu
論文名稱:NADH螢光半衰期與粒線體功能於STS誘導下細胞凋亡早期之相關性
論文名稱(外文):The Relationship of NADH Fluorescence Lifetime and Mitochondrial Function in the Early Stage of Staurosporine-Induced Apoptosis
指導教授:王興雯
指導教授(外文):Hsing-Wen Wang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生醫光電工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:67
中文關鍵詞:自體螢光半衰期細胞凋亡星形孢菌素
外文關鍵詞:NADH fluorescence lifetimeapoptosisstaurosporine
相關次數:
  • 被引用被引用:0
  • 點閱點閱:277
  • 評分評分:
  • 下載下載:16
  • 收藏至我的研究室書目清單書目收藏:0
我們先前的研究發現,細胞在1 �嵱 STS誘導細胞凋亡下,細胞NADH螢光半衰期立即顯著的增加。此螢光半衰期的增加主要是長時間的NADH螢光半衰期(也就是和蛋白質結合的NADH)成分的增加,顯示在STS誘導的凋亡下,NADH和蛋白質間的交互作用增加。由於此現象發生的時間早於caspase 3活化,結果亦建議也許NADH螢光半衰期的改變可以當成ㄧ個非侵入式方法早期偵測細胞凋亡的生物指標。細胞NADH螢光主要從粒線體而來,而STS誘導的細胞凋亡在caspase 3活化之前,更早期的現象發生在粒線體包括膜電位下降、細胞色素c的釋放、最終ATP下降等。這些現象和NADH螢光半衰期的變化尚未被研究、了解。本碩士論文進ㄧ步研究NADH螢光半衰期和粒線體功能之關係。我們先以低濃度(50 nM)Staurosporine(STS)的處理,caspase 3活化及細胞型態確定細胞走向凋亡, NADH螢光半衰期慢慢上升且半衰期變化最大值減少,顯示NADH螢光半衰期的變化速度及大小和STS藥物濃度成正比。接著,我們觀察粒線體缺陷的細胞在STS治療下,發現NADH螢光半衰期上升的變化隨粒線體的缺陷而增加,細胞亦加速凋亡。NADH螢光衰退時間的變化和粒線體膜電位及ATP於50nM及1�嵱 STS治療下的改變似乎沒有完全一樣的變化趨勢,但是有以下幾點關係: 1)NADH螢光衰退時間上升然後下降和膜電位於50 nM STS治療下的變化趨勢類似; 2) NADH螢光衰退時間上升都是發生在膜電位及ATP尚未下降之前; 3) NADH螢光衰退時間、膜電位及ATP最終都下降。最後,由於膜電位的上升反應粒線體可能喪失耗氧功能或電子傳遞鏈速度,從我們偵測細胞經STS處理後的耗氧速率,得知經藥物處理90分鐘後,粒線體的耗氧速率不變但粒線體的偶合作用降低。而耗氧在細胞及純粒線體分別於1及2小時有下降的趨勢。由此顯示NADH螢光半衰期的降低也許反應了粒線體的電子傳遞鏈及耗氧功能的缺失。
We have previously reported that cellular NADH fluorescence lifetime significantly increased immediately after 1 mM Staurosporine (STS) induced-apoptosis. The main contribution of this phenomenon is due to the increased amount and lifetime of the bound NADH component indicating increased interactions of NADH and various proteins during STS-induced apoptosis. This NADH lifetime increase took place before caspase 3 activation suggesting that it may be a potential biological marker of noninvasive early detection of apoptosis. Because the main NADH fluorescence is from mitochondria and in order to understand the origins of this NADH fluorescence lifetime increase, we studied its correlation with mitochondria functions including the change of mitochondria membrane potential (MMP) and ATP that are known “early” signatures of mitochondria mediated apoptosis before various caspase activation. The relationship between these phenomena and the change of NADH fluorescence lifetime has not been fully understood. In this study, we have performed time-course measurements of NADH fluorescence lifetime, MMP, ATP levels, as well as mitochondria coupling effects at high (1 mM) and low (50 nM) doses of STS induced apoptosis. Apoptosis was confirmed by both cell morphology change and caspase 3 activation NADH fluorescence lifetime was performed using two-photon fluorescence lifetime microscopy (FLIM) system at Image Core facility. The speed and the amount of the NADH fluorescence lifetime change is directly related to the concentration of STS (Fig. 1) that higher STS dose caused higher NADH lifetime change after treatment and within a fixed treatment time. In an additional study using various levels of mitochondrial dysfunction cells, higher NADH lifetime change with a fixed dose of STS treatment (100 nM) is correlated with severer mitochondrial dysfunction and earlier apoptosis (data now shown). Compared to MMP and ATP changes after low and high STS treatment of HeLa cells (Fig.3), NADH fluorescence lifetime did not seem to correlate with these mitochondrial-induced apoptosis signatures in time. However, several trends have been observed: 1) the rise and drop of the NADH fluorescence lifetime after treatment was similar to the MMP change; 2) this increase then decrease of the NADH fluorescence lifetime took place before the drop of MMP and ATP. Lastly, we have measured the respiratory function of mitochondria (Fig. 4) because the increase of MMP indicated the possibility of the respiratory function failure. However, we found that the mitochondrial respiratory function remains the same whereas its coupling was decreased after treatment of cells with 50 nM STS for 90 minutes and longer. Therefore, the increase then decrease of NADH fluorescence lifetime might reflect the beginning of mitochondrial dysfunction of HeLa cells and starting apoptotic pathway.
中文摘要 iv
Abstract v
縮寫表 vii
第一章 緒論 1
1-1螢光生命週期影像顯微術概論(Fluorescence Lifetime Image Microscopy , FLIM) 1
1-1-1 螢光生命週期影像顯微術 1
1-1-2 時間相關單光子計數 (Time Correlated Single Photon Counting, TCSPC) 2
1-1-3 儀器響應函數,二倍頻(Second Harmonic Generation, SHG) 2
1-2 NADH(Reduced Nicotinamide adenine dinucleotide) 3
1-3粒線體概論 4
1-4細胞凋亡 7
第二章 實驗材料與方法 11
2-1 細胞株 12
2-1-1 細胞質融合技術[32, 46] 12
2-2 細胞培養 13
2-3 緩衝溶液 13
2-4 玻片的前處理 14
2-5 細胞計數 14
2-6 偵測螢光半衰期 14
2-7 偵測NADH螢光半衰期 15
2-8 Caspase 3 活性分析 15
2-8-1 細胞蛋白質抽取 15
2-8-2 蛋白質定量 16
2-8-3活性測定 16
2-9 分析粒線體膜電位 16
2-10 偵測ATP含量 17
2-11 測量粒線體耗氧速率 18
2-12 粒線體DNA斷損含量檢測 19
2-12-1 DNA萃取 19
2-12-2 粒線體DNA 4,977 bp段損比例之定量 19
2-13統計分析 20
第三章 結果 22
3-1 HeLa cells於藥物處理後, NADH螢光半衰期的變化 22
3-2 Caspase 3 活性分析 23
3-3 cybrids於藥物處理後,NADH螢光半衰期�鄝的變化 23
3-4 粒線體膜電位的改變 24
3-5 全細胞(whole cell)ATP的總量 25
3-6 細胞(粒線體)的耗氧速率 26
3-7 NADH的螢光強度 26
第四章 討論 27
總結 31
參考資料 32
圖與表 40
1. J. R. Alcala, "The effect of harmonic conformational trajectories on protein fluorescence and lifetime distributions," The Journal of Chemical Physics 101, 4578 (1994).
2. J. R. Alcala, E. Gratton, and F. G. Prendergast, "Fluorescence lifetime distributions in proteins," Biophysical journal 51, 597-604 (1987).
3. K. Carlsson, and A. Liljeborg, "Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation," Journal of Microscopy 185, 37 (1997).
4. M. Straub, and S. W. Hell, "Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope," Applied Physics Letters 73, 1769 (1998).
5. J. B. Pawley, and B. R. Masters, "Handbook of biological confocal microscopy," Journal of Biomedical Optics 13, 029902 (2008).
6. W. Becker, and A. Bergmann, "Lifetime imaging techniques for optical microscopy," Becker & Hickl GmbH,. Berlin (2002).
7. T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, "Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections," Biophysical journal 91, 4665-4677 (2006).
8. P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, "Second-harmonic imaging microscopy of living cells," Journal of Biomedical Optics 6, 277 (2001).
9. N. Pollak, C. Dolle, and M. Ziegler, "The power to reduce: pyridine nucleotides–small molecules with a multitude of functions," Biochem. J 402, 205-218 (2007).
10. M. Ziegler, "Emerging roles of NAD in cellular signaling," Eur J Biochem 267, 1550-1564 (2000).
11. A. Gafni, and L. Brand, "Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase," Biochemistry 15, 3165-3171 (1976).
12. M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," Journal of Biomedical Optics 12, 024014 (2007).
13. A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, "Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species," Journal of Photochemistry & Photobiology, B: Biology 31, 101-112 (1995).
14. Y. Hatefi, "The mitochondrial electron transport and oxidative phosphorylation system," Annual review of biochemistry 54, 1015-1069 (1985).
15. D. A. Clayton, "Replication and transcription of vertebrate mitochondrial DNA," Annual review of cell biology 7, 453-478 (1991).
16. A. E. Senior, "ATP synthesis by oxidative phosphorylation," Am Physiological Soc 68, 177-231 (1988).
17. J. Sastre, F. V. Pallardo, J. G. de la Asuncion, and J. Vina, "Mitochondria, oxidative stress and aging," Free Radical Research 32, 189-198 (2000).
18. R. E. Giles, H. Blanc, H. M. Cann, and D. C. Wallace, "Maternal inheritance of human mitochondrial DNA," Proceedings of the National Academy of Sciences 77, 6715-6719 (1980).
19. N. Howell, P. F. Chinnery, S. S. Ghosh, E. Fahy, and D. M. Turnbull, "Transmission of the human mitochondrial genome," Human Reproduction 15, 235-245 (2000).
20. S. Anderson, A. T. Bankier, B. G. Barrell, M. H. L. De Bruijn, A. R. Coulson, J. Drouin, I. C. Eperon, D. P. Nierlich, B. A. Roe, and F. Sanger, "Sequence and organization of the human mitochondrial genome," Nature 290, 457-465 (1981).
21. C. Richter, J. W. Park, and B. N. Ames, "Normal oxidative damage to mitochondrial and nuclear DNA is extensive," Proceedings of the National Academy of Sciences 85, 6465-6467 (1988).
22. A. N. Ames, "Endogenous DNA damage as related to cancer and aging," Mutation research 214, 41-46 (1989).
23. Y. H. Wei, Y. S. Ma, H. C. Lee, C. F. Lee, and C. Y. Lu, "Mitochondrial theory of aging matures-roles of mtDNA mutation and oxidative stress in human aging," Zhonghua Yi Xue Za Zhi (Taipei) 64, 259-270 (2001).
24. D. C. Wallace, G. Singh, M. T. Lott, J. A. Hodge, T. G. Schurr, A. M. Lezza, L. J. Elsas 2nd, and E. K. Nikoskelainen, "Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy," Science 242, 1427-1430 (1988).
25. I. J. Holt, A. E. Harding, and J. A. Morgan-Hughes, "Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies," Nature 331(6158), 717-719 (1988).
26. A. M. Kogelnik, M. T. Lott, M. D. Brown, S. B. Navathe, and D. C. Wallace, "MITOMAP: a human mitochondrial genome database--1998 update," Nucleic acids research 26, 112-115 (1998).
27. J. Christodoulou, "Genetic defects causing mitochondrial respiratory chain disorders and disease," Hum Reprod 15, 28-43 (2000).
28. J. V. Leonard, and A. H. V. Schapira, "Mitochondrial respiratory chain disorders I: mitochondrial DNA defects," The Lancet 355, 299-304 (2000).
29. J. M. Shoffner, M. T. Lott, A. S. Voljavec, S. A. Soueidan, D. A. Costigan, and D. C. Wallace, "Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip-replication model and metabolic therapy," Proceedings of the National Academy of Sciences 86, 7952-7956 (1989).
30. E. A. Schon, R. Rizzuto, C. T. Moraes, H. Nakase, M. Zeviani, and S. DiMauro, "A direct repeat is a hotspot for large-scale deletion of human mitochondrial DNA," Science 244, 346-349 (1989).
31. J. V. Leonard, and A. H. V. Schapira, "Mitochondrial respiratory chain disorders II: neurodegenerative disorders and nuclear gene defects," The Lancet 355, 389-394 (2000).
32. Y. H. Wei, "Mitochondrial DNA mutations and oxidative damage in aging and diseases: an emerging paradigm of gerontology and medicine," Proceedings of the National Science Council, Republic of China. Part B, Life sciences 22, 55-67 (1998).
33. A. M. S. Lezza, P. Mecocci, A. Cormio, M. F. Beal, A. Cherubini, P. Cantatore, U. Senin, and M. N. Gadaleta, "Mitochondrial DNA 4977 bp deletion and OH8dG levels correlate in the brain of aged subjects but not Alzheimer's disease patients," FASEB 13, 1083-1088 (1999).
34. E. Finkel, "The mitochondrion: is it central to apoptosis?," Science 292(5517), 624-626 (2001).
35. E. A. Schon, and G. Manfredi, "Neuronal degeneration and mitochondrial dysfunction," Am Soc Clin Investig 111(3), 303-312 (2003).
36. D. L. Vaux, and A. Strasser, "The molecular biology of apoptosis," Proceedings of the National Academy of Sciences 93, 2239-2244 (1996).
37. P. Li, D. Nijhawan, I. Budihardjo, S. M. Srinivasula, M. Ahmad, E. S. Alnemri, and X. Wang, "Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade," Cell 91, 479-490 (1997).
38. H. Li, H. Zhu, C. J. Xu, and J. Yuan, "Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis," Cell 94, 491-502 (1998).
39. V. Adams, S. Gielen, R. Hambrecht, and G. Schuler, "Apoptosis in skeletal muscle with aging," Am J Physiol Regul Integr Comp Physiol 282, 519-527 ( 2002).
40. U. Svantesson, H. Takahashi, U. Carlsson, A. Danielsson, and K. Stibrant Sunnerhagen, "Muscle and tendon stiffness in patients with upper motor neuron lesion following a stroke," European journal of applied physiology 82, 275-279 (2000).
41. M. C. Monici, A. Toscano, P. Girlanda, O. Musumeci, and G. Vita, "Apoptosis in metabolic myopathies," Neuroreport 9, 2431 (1998).
42. S. W. Hetts, "To die or not to die An overview of apoptosis and its role in disease," Am Med Assoc 279, 300-307 (1998).
43. M. W. Karaman, S. Herrgard, D. K. Treiber, P. Gallant, C. E. Atteridge, B. T. Campbell, K. W. Chan, P. Ciceri, M. I. Davis, and P. T. Edeen, "A quantitative analysis of kinase inhibitor selectivity," Nature Biotechnology 26, 127 - 132 (2008).
44. S. Omura, Y. Iwai, A. Hirano, A. Nakagawa, J. Awaya, H. Tsuchiya, Y. Takahashi, and R. Asuma, "A new alkaloid AM-2282 of streptomyces origin taxonomy, fermentation, isolation and preliminary characterization," The Journal of Antibiotics 30, 275-282 (1977).
45. H. J. Chae, J. S. Kang, J. O. Byun, K. S. Han, D. U. Kim, S. M. Oh, H. M. Kim, S. W. Chae, and H. R. Kim, "Molecular mechanism of staurosporine-induced apoptosis in osteoblasts," Pharmacological Research 42, 373-381 (2000).
46. W. K. Porteous, A. M. James, P. W. Sheard, C. M. Porteous, M. A. Packer, S. J. Hyslop, J. V. Melton, C. Y. Pang, Y. H. Wei, and M. P. Murphy, "Bioenergetic consequences of accumulating the common 4977-bp mitochondrial DNA deletion," Eur J Biochem 257, 192-201 (1998).
47. M. Herrmann, H. M. Lorenz, R. Voll, M. Grunke, W. Woith, and J. R. Kalden, "A rapid and simple method for the isolation of apoptotic DNA fragments," Nucleic acids research 22, 5506-5507 (1994).
48. H. W. Wang, V. Gukassyan, C. T. Chen, Y. H. Wei, H. W. Guo, J. S. Yu, and F. J. Kao, "Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells," Journal of Biomedical Optics 13, 054011 (2008).
49. C. Y. I. Liu, C. F. Lee, C. Hong, and Y. Wei, "Mitochondrial DNA mutation and depletion increase the susceptibility of human cells to apoptosis," Annals of the New York Academy of Sciences 1011, 133-145 (2004).
50. A. W. Abu-Qare, and M. B. Abou-Donia, "Biomarkers of apoptosis: release of cytochrome c, activation of caspase-3, induction of 8-hydroxy-2'-deoxyguanosine, increased 3-nitrotyrosine, and alteration of p53 gene," Journal of toxicology and environmental health. Part B, Critical reviews 4, 313 (2001).
51. H. W. Guo, C. T. Chen, Y. H. Wei, O. K. Lee, V. Gukassyan, F. J. Kao, and H. W. Wang, "Reduced nicotinamide adenine dinucleotide fluorescence lifetime separates human mesenchymal stem cells from differentiated progenies," Journal of Biomedical Optics 13, 050505 (2008).
52. H. W. Wang, J. C. Finlay, K. Lee, T. C. Zhu, M. E. Putt, E. Glatstein, C. J. Koch, S. M. Evans, S. M. Hahn, and T. M. Busch, "Quantitative comparison of tissue oxygen and motexafin lutetium uptake by ex vivo and noninvasive in vivo techniques in patients with intraperitoneal carcinomatosis," Journal of Biomedical Optics 12, 034023 (2007).
53. C. Y. Liu, C. F. Lee, and Y. H. Wei, "Quantitative effect of 4977 bp deletion of mitochondrial DNA on the susceptibility of human cells to UV-induced apoptosis," Mitochondrion 7, 89-95 (2007).
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊