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研究生:鄭郁琦
研究生(外文):Yu-Chi Cheng
論文名稱:螢光劑參雜對生物擬細胞膜所造成影響之研究
論文名稱(外文):Modifications of model cell membrane physical properties due to the addition of fluorescent dyes
指導教授:施明智施明智引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物物理學研究所
學門:自然科學學門
學類:物理學類
論文種類:學術論文
畢業學年度:97
語文別:英文
論文頁數:88
中文關鍵詞:微脂粒螢光
外文關鍵詞:GUVfluorescence microscopy
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細胞膜的骨架是由於磷脂質分子組成的,因此由不同成份所構成的GUV (Giant Unilamellar Vesicle) 是研究細胞膜一個容易控制的系統。而為了追蹤GUV,我們用螢光光學顯微鏡,這也是細胞生物學常用來區分不同胞器的方法之一。在我們的研究當中,常用的生物染劑會明顯的改變GUV系統的相變條件與行為。從DPPC的等溫曲線中,我們知道DPPC有兩相:LC 跟gel phase。而這個相變化具有兩個特徵:面積密度的改變及班點的形成。在純DPPC構成的GUV系統的成分中添加1%的NBD螢光劑或是1%的RB螢光劑,會發現系統的相變過程有明顯的改變:含1%NBD的GUV系統在相變時會萎縮且相變溫度會比純DPPC的GUV系統更低,而含1%RB的GUV卻會脹破且相變溫度升高。這表示在GUV系統裡添加極微量的螢光劑在DPPC GUV裡看到明顯的不同。我們的結論是說如果一顆GUV要進行相變,那麼伴隨的體積變化則必然與通透性有關。NBD分子因為在親油的兩隻腳是非對稱的所以DPPC-NBD GUVs對水的通透性較好;而RB分子因為在親油的兩隻腳是對稱的因此DPPC-RB GUVs 對水的通透性較差。另一方面,NBD-DPPC GUVs跟RB-DPPC GUVs的相變溫度跟純DPPC GUVs比較則呈現相反的溫度變化。NBD-DPPC GUVs的相變溫度比純DPPC GUVs低;但是RB-DPPC GUVs的相變溫度比純DPPC GUVs高。我們的研究說明由於NBD-DPPC GUVs 有相分離所以會降低相變溫度。至於RB-DPPC GUVs,我們猜測因為複雜結構的形成所以造成相變溫度的提高。
Since phospholipids make up the backbone for cell membranes, a Giant Unilamellar Vesicle (GUV) composed of various lipids is a well-controlled system to study membranes. To track GUVs, we utilize fluorescence microscopy, also a common method in cell biology to distinguish cellular compartments. In this work, we found that pure DPPC GUVs observed with different fluorescent dyes have different transition behaviors. Isotherm of DPPC monolayers shows that pure DPPC has two distinct phases, LC phase and gel phase, and this phase transition is characterized by two observations: change in area density and domain formation. In GUVs incorporated with NBD-PC dye, GUV shrink at phase transition; while, DPPC GUVs visualized with Rhodamine-B dye burst before transition can be completed. We concluded that permeability to water is a dominant factor in considering whether or not a GUV can tolerate volume change thus completing phase transition. DPPC-GUVs incorporated with NBD allow better permeability to water due to the asymmetric hydrocarbon region of NBD; and DPPC-GUVs visualized with Rhodamine are less permeable to water because Rhodamine has symmetrical hydrocarbon tails like that of DPPC. On the other hand, the transition temperatures of DPPC-NBD and DPPC-RB GUVs exhibit opposite directional temperature shifts when compared with pure DPPC vesicles. The transition temperature for DPPC-NBD GUVs is lower than pure DPPC vesicle, while, transition temperature of DPPC-RB GUVs has a higher transition temperature than pure DPPC vesicles. In our study, we find that NBD-DPPC GUVs have phase separation and that brings down transition temperature. For RB-DPPC GUVs, we speculate that the forming complex structure be the factor responsible for raising transition temperature.
中文摘要 i
Abstract ii
Contents iii
Table Contents vi
Picture Contents vii
一、 Introduction 10
(一) Biological Membranes & Model cell Membranes 10
(二) Fluorescence Microscopy is a common method that tracks dynamic process 10
(三) Model Cell Membranes are bilayered vesicles 11
(四) Impurities additive to bilayered systems and monolayers 12
二、 Principles 23
(一) Plasma membrane 23
(二) Membrane Structure 23
(三) Lipid Fluidity 24
(四) Composition 24
(五) Bilayered Vesicles 25
(六) Vesicles Sizes 25
(七) Fluorescence probes 25
(八) Fluorescence microscopy 26
(九) Resolution 26
(一零) Phase-contrast microscopy 27
(一一) Monolayer 27
(一二) Isotherms 28
三、 Materials and Methods 33
(一) Materials 33
(二) Cleaning 33
(三) Sample Preparation 34
(四) Vesicle Incubation 35
(五) Temperature Determination 36
(六) Temperature-induced Phase transition 36
(七) Phase transition temperature and shrinking ratios calculation 37
(八) Isotherm 37
(九) CCD 38
四、 Results 44
(一) 4.1 Addition of 1% Fluorescence dye results in different DPPC phase transition phenomenon 44
(二) 4.2 Statistical counting for shrink and burst ratios 46
(三) 4.3 Different transition pattern originate from the same DPPC transition nature 47
(四) 4.4 Osmotic Pressure Testing 49
(五) 4.5 Comparison of phase transition temperature and area change 50
(六) 4.6 Effect of impurity amount 51
(七) 4.7 Charge Screening results in different phase transition behavior for DPPC-RG GUVs in pure water 53
(八) 4.8 Compare monolayers of DPPC-NBD and DPPC-RB 55
(九) 4.9 Adding RB dye disrupts the chiral domain in DPPC and DPPC-NBD monolayers 56
五、 Discussion 77
六、 Conclusions 85
七、 References 87
黃大維,國立中興大學論文,去氧核醣核酸分子與脂質分子單層膜及微脂粒作用之研究,2006
遲昭安,國立中興大學論文,FPTL誘發分子單層膜聚集現象的研究,2001
Molecular Biology of the Cell, 4th edition, 2002.
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Feride Severcan, Filiz. Effect of progesterone on DPPC membrane: Evidence for lateral phase separation and inverse action in lipid dynamics. Archives of Biochemistry and Biophysics 440 (2006): 141-47.
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Krasteva, N. Effect of Sugars and Dimethyl Sulfoxide on the Structure and Phase Behavior of DPPC Monolayers. Langmuir 17 (2001): 1209-214.
Marguet, Didier, Pierre-Francois Lenne, Herve Rigneault, and Hai-Tao He. Dynamics in the plasma membrane: how to combine fluidity and order. EMBO Journal 25 (2006): 3446-457.
McConlogue, Cary W., Daniel Malamud, and T. Kyle Vanderlick. Interaction of DPPC monolayers with soluble surfactants: electrostatic effects of membrane perturbants. Biochimica et Biophysica Acta 1372 (2006): 124-34.
Meer, Gerrit Van, Dennis R. Voelker, and Gerald W. Feigenson. "Membrane lipids: where they are and how they behave." Membrane lipids: where they are and how they behave 9 (2009): 114-24.
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Wojtowicz, K. The effect of 4-hydroxycoumarin and umbelliferone on DPPC bilayers. Ultrasound study. Eur. Phys. J. Special Topics 154 (2008): 285-88.
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