人造細胞膜的相行為及脂質-固醇交互作用之研究

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：78

、訪客IP：3.21.21.209

姓名

翁棋蓉(Chi-jung Weng) 查詢紙本館藏

畢業系所

物理學系

論文名稱

人造細胞膜的相行為及脂質-固醇交互作用之研究
(A study on the phase behavior and lipid-sterol interaction of model membranes)

相關論文

★ 用氘核磁共振儀研究含高濃度麥角脂醇的DPPC人造膜之分子交交互作用	★ Fluorescence study of lipid membranes containing sterol
★ 含固醇的脂質雙層膜的形態及相行為的研究	★ The effects of composition and thermal history on the properties of supported lipid bilayers
★ The effect of sterol on the POPE/DPPC membranes	★ 麥角固醇對含膽固醇的脂雙層膜的影響
★ Deuterium NMR Study of the Effect of Stigmasterol on POPE Membranes	★ Deuterium NMR Study of the effect of 7- dehydrocholesterol on the POPE Membranes
★ 運用氘核磁共振儀研究POPC/cholesterol膜之物理性質	★ 模型細胞膜(含有相同碳鏈的PC/PE)存在或缺乏固醇類的物理性質
★ 運用氘核磁共振研究DPPC/POPE/sterol人造細胞膜之物理性質	★ Phase Behavior and Molecular Interactions of Membranes Containing Phosphatidylcholines and Sterol: A Deuterium NMR Study
★ The physical properties of phytosterol-containing lipid bilayers	★ An AFM Study on Supported Lipid Bilayers with and without Sterol
★ β-谷固醇對POPE膜物理特性的影響	★ 固醇結構對PC膜物理特性的影響

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 ( 永不開放)

摘要(中)

固醇在生物膜的脂質組織及動力學性質扮演重要的角色。研究證實膜上富含膽固醇和磷鞘脂的脂筏區塊，在很多的生物功能的運作具有關鍵性的影響。然而，它們的存在和物理性質仍有爭議。為了對這些問題做深入的探討，我們研究飽和脂質、不飽和脂質、固醇組成的人造細胞膜的物理性質。研究中，我們運用螢光顯微術得到大型單層微胞(giant unilamellar vesicles)的形態隨溫度與成分的變化。由核磁共振(2H NMR)研究其人造細胞膜的相行為，進而建構該成分的相圖。結果顯示該三元混合物在相圖上不同的區域顯示不同的特性。例如，在固醇濃度較低的膠態與液態的共存區呈現微米尺度的小型分散的區塊(small, scattered domains)。而在固醇濃度較廣之液態-液態共存區,則形成微米尺度的大面積區塊(large domains)。
目前大多數研究聚焦於含低濃度(<30 mol%)固醇的脂質膜的探討，對於含高濃度固醇的脂質膜的研究相對較少。然而有些細胞膜中的膽固醇的濃度可以大於40 mol%。為了研究細胞膜中的高濃度固醇區域的物理性質，我們運用核磁共振光譜術與x-ray繞射研究脂質與固醇組成的人造細胞膜，我們發現相對於膽固醇分子結構，在碳氫鏈上額外的分子群與雙鍵，兩者皆阻礙了脂質與固醇之間的交互作用。另一方面，固醇環上額外的雙鍵對於脂質與固醇之間的交互作用並無影響。

摘要(英)

Sterols play important roles on lipid organization and dynamics in cell membranes. Rafts domains rich in cholesterol and sphingolipids have been implicated to play crucial roles in a wide range of biological processes. However, their existence and physical properties have been controversial. To gain a better understanding of these issues, we study the physical properties of model membranes composed of saturated lipid, unsaturated lipid and sterol. The morphology of giant unilamellar vesicles has been observed as a function of temperature and composition by fluorescence microscopy. The phase behavior of model membranes was investigated by deuterium nuclear magnetic resonance (2H NMR) spectroscopy. The composition phase diagram was constructed. This ternary mixture manifests itself differently at different regions of the phase diagram. Solid-liquid phase coexistence, displaying small, scattered domains in micron size, was observed at low sterol concentration. Liquid-liquid phase coexistence, exhibiting large domains in micron size, was observed in a wide sterol concentration range.
While most studies have been focused on membranes containing low sterol concentration (<30 mol %), less studies have been performed at the high sterol concentration. Cholesterol content can be as high as 40 mol % or greater in some cells. To investigate the physical properties of membranes at high sterol concentration regime, we study model membranes composed of lipid and sterol by 2H-NMR spectroscopy and x-ray diffraction. We found that in comparison with cholesterol, the extra group and extra double bond at the side chain of other sterols both hinder the lipid-sterol interaction. The extra double bond at the fuse ring, on the other hand, has no effect on the lipid-sterol interaction.

關鍵字(中)

★ 脂質
★ 固醇
★ 核磁共振
★ X光繞射

關鍵字(英)

★ lipid
★ sterol
★ 2H NMR
★ XRD

論文目次

中文摘要 I
ABSTRACT II
致謝 IV
圖表目錄 VIII
第一章緒論 1
1.1 細胞膜的結構 1
1.2 脂質分子的相行為（phase behavior） 3
1.3 三元混合的脂質膜之相關研究 5
1.4 脂質-固醇交互作用 11
第二章螢光顯微術 (Fluorescence Microscopy)及X-ray繞射 (X-ray Diffraction) 15
2.1 螢光顯微術 (Fluorescence Microscopy) 15
2.1.1 螢光發光原理 (Principle of Fluorescence) 15
2.1.2 螢光顯微鏡工作原理 16
2.1.3 螢光分子 19
2.1.4 實驗材料 21
2.1.5 GUVs的製備 22
2.2 X-Ray繞射 (XRD) 24
2.2.1 X-ray繞射基本原理 24
2.2.2 實驗材料 28
2.2.3 XRD實驗樣品MLVs的製備 28
2.2.4 X-ray繞射數據量測 29
第三章核磁共振光譜術 (2H NMR Spectroscopy) 30
3.1 NMR原理 30
3.2 電四極交互作用(Quadrupole Interaction) 32
3.3 相行為(Phase Behavior)及First Moment M1 35
3.4 實驗材料 37
3.5 2H NMR實驗樣品MLVs的製備 37
3.6 2H NMR光譜的量測 38
第四章 POPC/DPPC/erg脂質膜之形態與相行為之研究 39
4.1 GUVs形態之簡介 39
4.2 POPC/DPPC脂質膜的形態及相行為 44
4.2.1 螢光實驗與2H NMR量測的相變溫度之比較 44
4.2.2 POPC/DPPC脂質膜之形態及相行為隨成分的變化 45
4.3 POPC/DPPC/erg脂質膜之形態及相行為隨成分的變化 48
4.4 POPC/DPPC/erg相圖 56
4.5 POPC/DPPC/erg脂質膜形態及相行為隨溫度的變化 60
第五章固醇與POPE交互作用之探討 64
5.1 Pure POPE脂質膜 64
5.2 POPE/chol脂質膜 68
5.3 POPE/erg脂質膜 76
5.4 POPE/β-sitosterol脂質膜 81
5.5 POPE/stigmasterol脂質膜 86
5.6 固醇對脂質膜溶解差異探討 91
第六章結論 95
參考文獻 99

參考文獻

[1] Singer S. J. and Garth L. Nicolson. 1972. The Fluid Mosaic Model of the Structure of Cell Membranes. Science 175: 720-731.
[2] D. A. Brown and E. London. 1998. Functions of Lipid Rafts in Biological membranes. Annu. Rev. Cell Dev. Biol. 14:111-36.
[3] D. A. Brown and E. London. (2000). Structure and Function of Sphingolipid- and Cholesterol-rich Membrane Rafts. J. Biol. Chem. 275: 17221-17224.
[4] A. T. Hammond, F. A. Heberle,…, G. W. Feigenson. 2005. Crosslinking a lipid raft component triggers liquid ordered–liquid disordered phase separation in model plasma membranes. Proc Natl Acad Sci USA 102: 6320-6325.
[5] Linda J. Pike. 2003. Lipid rafts: bringing order to chaos. J Lipid Res 44: 655-667.
[6] Kai Simons and Elina Ikonen. 1997. Functional rafts in cell membranes. Nature 387: 569-572.
[7] Kai Simons and Robert Ehehalt. 2002. Cholesterol, lipid rafts, and disease. J. Clin. Invest. 110: 597-603.
[8] Marc Fivaz, Laurence Abrami, and F.Gisou van der Goot. 1999. Landing on lipid raft. Trends Cell Biol. 9: 212-213.
[9] Philip L. Yeagle. 2004 The structure of biological membranes, 2nd edition. CRC Press LLC, Boca Raton, p. 26.

[10] J. Rubenstein, B.A. Smith, and H.M. McConnell. 1979. Lateral diffusion in binary mixtures of cholesterol and phosphatidylcholines. Proc. Natl. Acad. Sci. 76: 15-18.
[11] Sean Munro. Lipid Rafts: Elusive or Illusive?. 2003 Cell 115: 377-388.
[12] Margus R. Vist and James H. Davis. 1990. Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic resonance and differential scanning calorimetry. Biochemistry. 29: 451-464.
[13] Ya-Wei Hsueh, Kyle Gilbert, …, Jenifer Thewalt. 2005. The effect of ergosterol on dipalmitoylphosphatidylcholine (DPPC) bilayers: a deuterium NMR and calorimetric study. Biophys. J. 88: 1799-1808.
[14] Ya-Wei Hsueh, Mei-Ting Chen, …, Jenifer Thewalt. 2007. Ergosterol in POPC Membranes: Physical Properties and Comparison with Structurally Similar Sterols. Biophys. J. 92: 1606-1615.
[15] Ruiguang Wu, Lin Chen, …, Peter J. Quinn. 2006. Phase diagram of stigmasterol/dipalmitoylphosphatidylcholine mixtures dispersed in excess water. Biochimica et Biophysica Acta. 1758: 764-771.
[16] Pietro Cicuta, Sarah L. Keller and Sarah L. Veatch. 2007. Diffusion of Liquid Domains in Lipid Bilayer Membranes. J. Phys. Chem. B. 111: 3328-3331
[17] Sarah L. Veatch and Sarah L. Keller. 2002 Organization in Lipid Membranes Containing Cholesterol. PRL. 89: 268101-1-4.
[18] Sarah L. Veatch and Sarah L. Keller. (2005). Miscibility phase diagrams of giant vesicles containing sphingomyelin. Phys. Rev. Lett. 94: 148101.
[19] Jiang Zhao, Jing Wu, …, Gerald W. Feigenson. 2007. Phase studies of model biomembranes: Complex behavior of DSPC/DOPC/Cholesterol. Biochimica et Biophysica Acta. 1768: 2764-2776.
[20] Rodrigo F. M. de Almeida, Aleksandre Fedorov, and Manuel Prieto. 2003. Sphingomyelin/Phosphatidylcholine/Cholesterol Phase Diagram: Boundaries and Composition of Lipid Rafts. Biophys. J. 85: 2406-2416.
[21] Gerald W. Feigenson and Jeffrey T. Buboltz. 2001. Ternary Phase Diagram of Dipalmitoyl-PC/Dilauroyl-PC/Cholesterol: Nanoscopic Domain Formation Driven by Cholesterol. Biophys. J. 80: 2775-2788.
[22] Sarah L. Veatch, Klaus Gawrisch, and Sarah L. Keller. 2006. Closed-Loop Miscibility Gap and Quantitative Tie-Lines in Ternary Membranes Containing Diphytanoyl PC. Biophys. J. 90: 4428-4436.
[23] Robert B. Gennis. 1989. Biomembranes: molecular structure and function. Springer-Verlag. P. 22.
[24] http://avantilipids.com/
[25] Ajuna Arora, H. Raghuraman, and Amitabha Chattopadhyay. 2004. Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach. Biochemical and Biophysical Research Communications. 318: 920-926.
[26] Rie Bak J€apelt, Thomas Didion, …, Jette Jakobsen. 2011. Seasonal Variation of Provitamin D2 and Vitamin D2 in Perennial Ryegrass (Lolium perenne L.). |J. Agric. Food Chem. 59: 10907-10912.
[27] B.D. Ladbrooke, R.M. Williams, D. Chapman. 1968. Studies on lecithin–cholesterol–water interactions by differential scanning calorimetry and X-ray diffraction. Biochimica et Biophysica Acta. 150: 333-340.
[28] Wen Guo and James A. Hamilton. 1995 A multinuclear solid-state NMR study of phospholipid–cholesterol interactions dipalmitoyl phosphatidylcholine–cholesterol binary system. Biochemistry. 34: 14174-14184.
[29] John J. Collins and Michael C. Phillips. 1982. The stability and structure of cholesterolrich codispersions of cholesterol and phosphatidylcholine J. Lipid Res. 23: 291-298.
[30] Knoll W, Schmidt G, Ibel K, Sackmann E. 1985. Small-angle neutron scattering study of lateral phase separation in dimyristoylphosphatidylcholine-cholesterol mixed membranes. Biochemistry 24: 5240-5246.
[31] Michael R. Brzustowicz, Vadim Cherezov, …, Stephen R. Wassall. 2002. Molecular Organization of Cholesterol in Polyunsaturated Membranes: Microdomain Formation. Biophys. J. 82: 285-298.
[32] Aden Hodzic, Michael Rappolt, …, Georg Pabst. 2008. Differential Modulation of Membrane Structure and Fluctuations by Plant Sterols and Cholesterol. Biophys. J. 94: 3935–3944.
[33] Mark M. Stevens, Aurelia R. Honerkamp-Smith and Sarah L. Keller. 2010. Solubility limits of cholesterol, lanosterol, ergosterol, stigmasterol, and β-sitosterol in electroformed lipid vesicles. Soft Matter. 6: 5882-5890.

[34] James J. Cheetham, Ellen Wachtel, …, Richard M. Epand. 1989 Role of the Stereochemistry of the Hydroxyl Group of Cholesterol and the Formation of Nonbilayer Structures in Phosphatidylethanolamines. Biochemistry 28: 8928-8934.
[35] Z. Chen and R. P. Rand. 1997. The influence of cholesterol on phospholipid membrane curvature and bending elasticity. Biophys. J. 73: 267-276.
[36] Juyang Huang and Gerald W. Feigenson. 1999. A Microscopic Interaction Model of Maximum Solubility of Cholesterol in Lipid Bilayers. Biophys. J. 76: 2142-2157.
[37] Aden Hodzic, Michael Rappolt,…, Georg Pabst. 2008. Differential Modulation of Membrane Structure and Fluctuations by Plant Sterols and Cholesterol. Biophys. J. 94: 3935–3944.
[38] Piotr Zabrocki, , Ilse Bastiaens, …, Fred Van Leuven, and Joris Winderickx. 2008. Phosphorylation, lipid raft interaction and traffic of α-synuclein in a yeast model for Parkinson. Biochimica et Biophysica Acta 1783: 1767-1780.
[39] Chun Shi, Fengming Wu, …, Jie Xu. 2013. Incorporation of β-sitosterol into the membrane increases resistance to oxidative stress and lipid peroxidation via estrogen receptor-mediated PI3K/GSK3β signaling. Biochimica et Biophysica Acta 1830: 2538-2544.
[40] Markus Veen, Ulf Stahl, Christine Lang. 2003. Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae. FEMS Yeast Research 4: 87-95.
[41] Tomoko Iwaki, Haruyuki Iefuji, …, Yuko Giga-Hama, and Kaoru Takegawa. 2008. Multi ergosterol biosynthetic pathway leads to accumulation of yeast Schizosaccharomyces pombe. Microbiology. 154: 830-841.
[42] Paulo F. F. Almeida, Winchil L. C. Vaz, T. E. Thompson. 1992. Lateral diffusion in the liquid phases of dimyristoylphosphatidylcholine/ cholesterol lipid bilayers: a free volume analysis. Biochemistry. 31: 6739–6747.
[43] Katrin K. Halling, and J. Peter Slotte. 2004. Membrane properties of plant sterols in phospholipid bilayers as determined by differential scanning calorimetry, resonance energy transfer and detergent-induced solubilization. Biochimica et Biophysica Acta. 1664: 161-171.
[44] Ann M. Lees, Henry Y.I. Mok, …, Martha A. McCluskey, and Scott M. Grundy. 1977. Plant sterols as cholesterol-lowering agents: clinical trials in patients with hypercholesterolemia and studies of sterol balance. Atherosclerosis. 28: 325-338.
[45] Bradford, P. G., and A. B. Awad. 2007. Phytosterols as anticancer compounds. Mol. Nutr. Food Res. 51: 161-170.
[46] http://www.sigmaaldrich.com/taiwan.html
[47] Albani, J.R., Structure and dynamics of macromolecules: absorption and fluorescence studies, Elsevier 2004
[48] http://zeiss-campus.magnet.fsu.edu/articles/lightsources/mercuryarc.html.

[49] http://nikon2.magnet.fsu.edu/articles/fluorescence/filtercubes/blue/b2a/b2aindex.html
[50] http://avantilipids.com/index.php?option=com_content&view=article&id=1013&Itemid=215&catnumber=810145.
[51] Rodrigo F. M. de Almeida, JanWillem Borst, …, Antonie J. W. G. Visser. 2007. Complexity of lipid domains and rafts in giant unilamellar vesicles revealed by combining imaging and microscopic and macroscopic time-resolved fluorescence. Biophys. J. 93: 539-553.
[52] Tobias Baumgart, Geoff Hunt,…, Gerald W. Feigenson. 2007. Fluorescence probe partitioning between Lo/Ld phases in lipid membranes. Biochim Biophys Acta. 1768: 2182–2194.
[53] L. Loura, R.F.M. De Almeida, L.C. Silva, M. Prieto. 2009. FRET analysis of domain formation and properties in complex membrane systems. Biochim Biophys Acta. 1788 : 209-224.
[54] R.F.M. de Almeida, L. Loura, M. Prieto. 2009. Membrane lipid domains and rafts: current applications of fluorescence lifetime spectroscopy and imaging. Chemistry and physics of lipids. 157: 61-77.
[55] Veronika Kralj-Iglič, Gregor Gomišček,…, and Saša Svetina. 2001. Myelin-like protrusions of giant phospholipid vesicles prepared by electroformation. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 181: 315–318.
[56] S. L. Veatch, I. V. Polozov, K. Gawrisch, and S. L. Keller. 2004. Liquid domains in vesicles investigated by NMR and fluorescence microscopy. Biophys. J. 86: 2910-2922.
[57] L. A. Bagatolli and E. Gratton. 2000. Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures. Biophys. J. 78: 290-305.
[58] Kirsten Bacia, Petra Schwille, and Teymuras Kurzchalia. 2005. Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes. Proc. Natl. Acad. Sci. 102: 3272-3277.
[59] Riqiang Fu and Timothy A. Cross. 1999. Solid-state nuclear magnetic resonance investigation of protein and polypeptide structure. Annu Rev Biophys Biomol Struct. 28:235-68.
[60] Maria Victoria Silva Elipe. 2003. Advantages and disadvantages of nuclear magnetic resonance spectroscopy as a hyphenated technique. Analytica Chimica Acta 497: 1–25.
[61] David F. Bocian and Sunney I. Chan. 1978. NMR studies of membrane structure and dynamics. Ann. Rev. Phys. Chem. 29: 307-35.
[62] Michael F. Brown, Silvia Lope-Piedrafita,..., Horia I. Petrache. 2006. Solid-State Deuterium NMR Spectroscopy of Membranes. Springer.
[63] David B. Fenske and Pieter R. Cullis. 1995. Lipid Polymorphism. John Wiley & Sons, New York, pp. 2730-2735.
[64] J. Henriksen, A. C. Rowat,…, J. H. Ipsen. 2006. Universal Behavior of Membranes with Sterols. Biophys. J. 88:1799-1808.
[65] Davis, J. H., K. R. Jeffrey, …, T. P. Higgs. 1976. Quadrupolar echo deuteron magnetic resonance spectroscopy in ordered hydrocarbon chains. Chem. Phys. Lett. 42:390–394.
[66] Chi-Jung Weng and Ya-Wei Hsueh. Unpublished data.
[67] Ya-Wei Hsueh, Kyle Gilbert,…, Jenifer Thewalt. 2005. The Effect of Ergosterol on Dipalmitoylphosphatidylcholine Bilayers: A Deuterium NMR and Calorimetric Study. Biophys. J. 88: 1799-1808.
[68] Ming- Yen, Kuo and Ya-Wei Hsueh. 2009. Phase Behavior and Molecular Interaction of Membranes Containing Phosphatidylcholines and Sterol: A Deuterium NMR Study. M.S. thesis.
[69] Ju-Ping Wu and Ya-Wei Hsueh. 2009. Fluorescence study of lipid membranes containing sterol. M.S. thesis.
[70] Sarah L. Veatch. 2007. From small fluctuations to large-scale phase separation: Lateral organization in model membranes containing cholesterol. Seminars in cell & developmental biology 18: 573–582.
[71] W. Patrick Williams, Beth A. Cunningham, …, Wim Bras. 1996. A combined SAXS/WAXS investigation of the phase behaviour of di-polyenoic membrane lipids. Biochimica et Biophysica Acta. 1284: 86-96.
[72] B.D. Ladbrooke, R.M. Williams, D. Chapman. 1968 Studies on lecithin–cholesterol – water interactions by differential scanning calorimetry and X-ray diffraction, Biochim. Biophys. Acta 150: 333-340.
[73] Richard M. Epand, Diana Bach,…, Ellen Wachtel. 2000. Cholesterol crystalline polymorphism and the solubility of cholesterol in phosphatidylserine. Biophys. J. 78: 866–873.
[74] Juyang Huang, Je¡rey T. Buboltz and Gerald W. Feigenson. 1999. Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers. Biochimica et Biophysica Acta. 1417: 89-100.
[75] B.M. Craven. 1976 Crystal structure of cholesterol monohydrate. Nature 260: 727-729.
[76] Diana Bach and Ellen Wachtel. 2003. Phospholipid/cholesterol model membranes: formation of cholesterol crystallites. Biochimica et Biophysica Acta 1610: 187-197.
[77] Richard S. Abendan and Jennifer A. Swift. 2002. Surface Characterization of Cholesterol Monohydrate Single Crystals by Chemical Force Microscopy. Langmuir. 18: 4847-4853.
[78] Wei-Chin Hung, Ming-Tao Lee, …, Huey W. Huang. 2007. The Condensing Effect of Cholesterol in Lipid Bilayers. Biophys. J. 92: 3960-3967.
[79] Ya-Wei Hsueh, Chi-Jung Weng, …, Martin Zuckermann. 2010. Deuterium NMR Study of the Effect of Ergosterol on POPE Membranes. Biophys. J. 98: 1209-1217.

指導教授

薛雅薇(Ya-wei Hsueh)

審核日期

2016-1-27

推文