在本實驗中利用螢光相關光譜測量脂質體 DOPC 的膜流動性，配合拉曼光譜測量脂 質體 DOPC 的結構變化。實驗中加入過氧化氫對於脂質體 DOPC 膜的流動性與結構沒 有明顯的變化；加入氫氧自由基後對於 DOPC 的流動性有顯著的增快情形，同時觀察結 構變化也發現脂質體的雙鍵有受到破壞的現象。我們推論是因為脂質受到氧化之後造成 碳鏈變短，使得流動性變快。
在實驗中也分別在脂質體加入了水溶性抗氧化劑維他命 C 和脂質中嵌入脂溶性抗 氧化劑維他命 E，這兩種抗氧化劑皆屬於阻斷氧化反應持續生成的抗氧化劑。從實驗結 果得到含有抗氧化劑的脂質體對於加入氫氧自由基造成流動性變快的現象，有明顯抑制 的結果，脂質雙鍵被破壞的情形也有減緩，確實脂質體是因為發生氧化造成結構破壞與 流動性增快。
為了讓脂質體模型更接近真實細胞膜，我們在脂質體膜上也嵌入不同比例的膽固 醇。含有膽固醇的脂質體 DOPC 在膜流動性上有隨著膽固醇比例增加而減慢；加入了氫 氧自由基後含有少量膽固醇的脂質體 DOPC 還是有膜流動性增快的現象。但是在含 32.9%膽固醇的脂質體 DOPC 卻沒有變快；從拉曼光譜的結果在每種含膽固醇的脂質體 DOPC 皆有脂質雙鍵被破壞的情形。我們推斷是大部分氫氧自由基是破壞脂質雙鍵，僅 有少量受到氫氧自由基破壞膽固醇結構，然而在高比例膽固醇存在的時候，大部分膽固 醇沒有被破壞所以還是有維持細胞膜結構的功能存在，穩固了脂質體 DOPC 因為氫氧自 由基的攻擊造成的流動速度變快現象。
我們利用了螢光相關光譜和拉曼光譜推論出了脂質過氧化膜流動性與結構變化之 間的關係，未來將可以使用這次實驗所得到的技術，研究其他不同模型。真實細胞上含
參考文獻
1. Oghalai, J.S., Zhao, H.-B., Kutz, J.W. & Brownell, W.E. Voltage- and tension-dependent lipid mobility in the outer hair cell plasma membrane. Science 287, 658-661 (2000).
2. Tulenko, T.N., Chen, M., Mason, P.E. & Mason, R.P. Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis. Journal of Lipid Research 39, 947-956 (1998).
3. Ishitsuka, Y., Pham, D.S., Waring, A.J., Lehrer, R.I. & Lee, K.Y.C. Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: Effect of head group electrostatics and tail group packing. Biochimica Et Biophysica Acta-Biomembranes 1758, 1450-1460 (2006).
4. Garcia-Saez, A.J. & Schwille, P. Fluorescence correlation spectroscopy for the study of membrane dynamics and protein/lipid interactions. Methods (2008).
5. Wayne M. Becker, L.J.K., Jeff Hardin The World of the Cell, Edn. 6th. Benjamin Cummings, 2005.
6. Vereb, G. et al. Dynamic, yet structured: The cell membrane three decades after the Singer-Nicolson model. Proceedings of the National Academy of Sciences of the United States of America 100, 8053-8058 (2003).
7. Marguet, D., Lenne, P.F., Rigneault, H. & He, H.T. Dynamics in the plasma membrane:
how to combine fluidity and order. Embo Journal 25, 3446-3457 (2006).
8. 王鏡岩、朱聖庚、徐長法，生物化學，藝軒圖書出版社，2007 年 4 月。
11. Tocanne, J.F., Dupoucezanne, L., Lopez, A. & Tournier, J.F. Lipid lateral diffusion and membrane organization. Febs Letters 257, 10-16 (1989).
12. Christian Le Grimellec, Gerard Friedlander, El Hossain El Yandouzi, Philippe Zlatkine
& Giocondi, M.-C. Membrane fluidity and transport properties in epithelia. Kidney International 42, 825-826 (1992).
13. Becker, L.B. New concepts in reactive oxygen species and cardiovascular reperfusion physiology. Cardiovascular Research 61, 461-470 (2004).
14. Matsumi, Y., Kono, M., Ichikawa, T., Takahashi, K. & Kondo, Y. Laser-induced fluorescence instrument for the detection of tropospheric OH radicals. Bulletin of the Chemical Society of Japan 75, 711-717 (2002).
15. Naziroglu, M. New molecular mechanisms on the activation of TRPM2 channels by oxidative stress and ADP-ribose. Neurochemical Research 32, 1990-2001 (2007).
16. Winterbourn, C.C. Reconciling the chemistry and biology of reactive oxygen species.
Nature Chemical Biology 4, 278-286 (2008).
17. Ingraham, L.M., Boxer, L.A., Haak, R.A. & Baehner, R.L. Membrane fluidity changes accompanying phagocytosis in normal and in chronic granulomatous-disease polymorphonuclear leukocytes. Blood 58, 830-835 (1981).
18. Marnett, L.J. Lipid peroxidation - DNA damage by malondialdehyde. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis 424, 83-95 (1999).
19. Choe, M., Jackson, C. & Yu, B.P. Lipid-peroxidation contributes to age-related membrane rigidity. Free Radical Biology and Medicine 18, 977-984 (1995).
22. Verstraeten, S.V., Nogueira, L.V., Schreier, S. & Oteiza, P.I. Effect of trivalent metal ions on phase separation and membrane lipid packing: Role in lipid peroxidation.
Archives of Biochemistry and Biophysics 338, 121-127 (1997).
23. Garcia, J.J. et al. Melatonin enhances tamoxifen's ability to prevent the reduction in microsomal membrane fluidity induced by lipid peroxidation. Journal of Membrane Biology 162, 59-65 (1998).
24. Borst, J.W., Visser, N.V., Kouptsova, O. & Visser, A. Oxidation of unsaturated phospholipids in membrane bilayer mixtures is accompanied by membrane fluidity changes. Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids 1487, 61-73 (2000).
25. Gazawi, H. et al. The effects of bile acids on beta-adrenoceptors, fluidity, and the extent of lipid peroxidation in rat cardiac membranes. Biochemical Pharmacology 59, 1623-1628 (2000).
26. Chen, L.J., Yang, X.Q., Jiao, H.L. & Zhao, B.L. Tea catechins protect against lead-induced cytotoxicity, lipid peroxidation, and membrane fluidity in HepG2 cells.
Toxicological Sciences 69, 149-156 (2002).
27. Cazzola, R., Rondanelli, M., Russo-Volpe, S., Ferrari, E. & Cestaro, B. Decreased membrane fluidity and altered susceptibility to peroxidation and lipid composition in overweight and obese female erythrocytes. Journal of Lipid Research 45, 1846-1851 (2004).
28. Yoshimoto, M. et al. Phosphatidylcholine vesicle-mediated decomposition of hydrogen peroxide. Langmuir 23, 9416-9422 (2007).
29. Folmer, V. et al. H O induces rapid biophysical and permeability changes in the
169-178 (1981).
31. Sergent, O. et al. Role for membrane fluidity in ethanol-induced oxidative stress of primary rat hepatocytes. Journal of Pharmacology and Experimental Therapeutics 313, 104-111 (2005).
32. Nourissat, P. et al. Ethanol induces oxidative stress in primary rat hepatocytes through the early involvement of lipid raft clustering. Hepatology 47, 59-70 (2008).
33. Christova, Y., James, P.S. & Jones, R. Lipid diffusion in sperm plasma membranes exposed to peroxidative injury from oxygen free radicals. Molecular Reproduction and Development 68, 365-372 (2004).
34. Fushimi, K., Dix, J.A. & Verkman, A.S. Cell-membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging.
Biophysical Journal 57, 241-254 (1990).
35. Zubenko, G.S., Kopp, U., Seto, T. & Firestone, L.L. Platelet membrane fluidity individuals at risk for Alzheimer's disease: a comparison of results from fluorescence spectroscopy and electron spin resonance spectroscopy. Psychopharmacology 145, 175-180 (1999).
36. Fukaya, T. et al. Arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats. Neurobiology of Aging 28, 1179-1186 (2007).
37. Chen, Y., Lagerholm, B.C., Yang, B. & Jacobson, K. Methods to measure the lateral diffusion of membrane lipids and proteins. Methods 39, 147-153 (2006).
38. Day, C.A. & Kenworthy, A.K. Tracking microdomain dynamics in cell membranes.
Biochimica Et Biophysica Acta-Biomembranes 1788, 245-253 (2009).
41. Rigler, R., Mets, U., Widengren, J. & Kask, P. Fluorescence correlation spectroscopy with high count rate and low-background - analysis of translational diffusion.
European Biophysics Journal with Biophysics Letters 22, 169-175 (1993).
42. Berland, K.M., So, P.T.C. & Gratton, E. Two-photon fluorescence correlation spectroscopy - method and application to the intracellular environment. Biophysical Journal 68, 694-701 (1995).
43. Schwille, P., MeyerAlmes, F.J. & Rigler, R. Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophysical Journal 72, 1878-1886 (1997).
44. Heinze, K.G., Rarbach, M., Jahnz, M. & Schwille, P. Two-photon fluorescence coincidence analysis: Rapid measurements of enzyme kinetics. Biophysical Journal 83, 1671-1681 (2002).
45. Petersen, N.O. Scanning fluorescence correlation spectroscopy .1. Theory and simulation of aggregation measurements. Biophysical Journal 49, 809-815 (1986).
46. Petersen, N.O., Hoddelius, P.L., Wiseman, P.W., Seger, O. & Magnusson, K.E.
Quantitation of membrane-receptor distributions by image correlation spectroscopy - concept and application. Biophysical Journal 65, 1135-1146 (1993).
47. Thompson, N.L., Burghardt, T.P. & Axelrod, D. Measuring surface dynamics of biomolecules by total internal-reflection fluorescence with photobleaching recovery or correlation spectroscopy. Biophysical Journal 33, 435-454 (1981).
48. Kastrup, L., Blom, H., Eggeling, C. & Hell, S.W. Fluorescence fluctuation spectroscopy in subdiffraction focal volumes. Physical Review Letters 94 (2005).
49. Elson, E.L., Schlessinger, J., Koppel, D.E., Axelrod, D. & Webb, W.W. Measurement
51. Korlach, J., Schwille, P., Webb, W.W. & Feigenson, G.W. Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy.
Proceedings of the National Academy of Sciences of the United States of America 96, 8461-8466 (1999).
52. Schwille, P., Haupts, U., Maiti, S. & Webb, W.W. Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophysical Journal 77, 2251-2265 (1999).
53. Bacia, K., Majoul, I.V. & Schwille, P. Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis. Biophysical Journal 83, 1184-1193 (2002).
54. Haupts, U., Maiti, S., Schwille, P. & Webb, W.W. Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. Proceedings of the National Academy of Sciences of the United States of America 95, 13573-13578 (1998).
55. Kettling, U., Koltermann, A., Schwille, P. & Eigen, M. Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proceedings of the National Academy of Sciences of the United States of America 95, 1416-1420 (1998).
56. Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 26 (2009).
57. Krichevsky, O. & Bonnet, G. Fluorescence correlation spectroscopy: the technique and its applications. Reports on Progress in Physics 65, 251-297 (2002).
chromosomes - sample degradation occurs with 514.5nm but not with 660nm laser-light. Experimental Cell Research 195, 361-367 (1991).
61. Angelova, M.I. & Dimitrov, D.S. Liposome electroformation. Faraday Discussions 81, 303-311 (1986).
62. Halliwell, B. & Whiteman, M. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? British Journal of Pharmacology 142, 231-255 (2004).
63. Van den Bogaart, G., Krasnikov, V. & Poolman, B. Dual-color fluorescence-burst analysis to probe protein efflux through the mechanosensitive channel MscL.
Biophysical Journal 92, 1233-1240 (2007).
64. Benda, A. et al. How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy. Langmuir 19, 4120-4126 (2003).
65. 郭寶錚、陳玉敏，生物統計入門，五南圖書出版股份有限公司，2007。
66. Movasaghi, Z., Rehman, S. & Rehman, I.U. Raman spectroscopy of biological tissues.
Applied Spectroscopy Reviews 42, 493-541 (2007).
67. Arora, A., Byrem, T.M., Nair, M.G. & Strasburg, G.M. Modulation of liposomal membrane fluidity by flavonoids and isoflavonoids. Archives of Biochemistry and Biophysics 373, 102-109 (2000).
68. Gabbianelli, R., Cifani, C., Massi, M., Polidori, C. & Falcioni, G. Oxidative damage in rat erythrocyte membranes following ethanol intake: Effect of ethyl pyruvate.
Chemico-Biological Interactions 169, 122-131 (2007).
69. Kahya, N. & Schwille, P. How phospholipid-cholesterol interactions modulate lipid
Physics of Lipids 146, 43-53 (2007).