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標題: 螢光劑參雜對生物擬細胞膜所造成影響之研究
Modifications of model cell membrane physical properties due to the addition of fluorescent dyes
作者: 鄭郁琦
Cheng, Yu-Chi
Contributors: 施明智
中興大學
關鍵字: GUV;fluorescence microscopy
微脂粒;螢光
日期: 2009
Issue Date: 2012-09-04 09:15:34 (UTC+8)
Publisher: 生物物理學研究所
摘要: 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.
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