- 學位論文
微米尺度的隔離式及懸吊式平面磷脂雙層膜的製備與生物應用
The Preparation And Biological Application Of Microsize Isolated And Suspended Planar Lipid Bilayers
摘要
支撐型磷脂雙層膜(Supported lipid bilayer)是一種簡化平面人工細胞膜，應用於研究蛋白質與模擬細胞膜的作用。一般使用囊泡融合(vesicle fusion)方法製備。但由於其膜面積太大不易使用於螢光漂白回填實驗，且與基材間只有1奈米水層，對於研究穿透磷脂膜蛋白而言，1奈米水層限制蛋白質穿透部分的結構，而影響蛋白質的功能。另外，由於負電膜電性跟玻璃表面的負電相斥，所以製備上較困難。具流動性且隔離的磷脂雙層膜較能方便觀察蛋白質與細胞膜不同排列區域的作用情形。本實驗室曾使用刮除法及氣泡剝離法在磷脂雙層膜上形成隔離區塊。但此兩種方法以破壞細胞膜來形成隔離式磷脂雙層膜，其隔離的細胞膜邊緣結構殘缺不全，難以知道其排列情形。 本研究使用氣泡崩解沉降法(bubble collapse deposition)製備隔離式磷脂雙層膜。其方法是利用氣泡吸取水中磷脂，然後將氣泡移至乾淨玻璃上並緩慢抽掉空氣而形成的磷脂雙層膜。此方法製備的磷脂膜邊緣是由磷脂自然排列形成而非由急速破壞造成，所以情形單純。另外，也研究製備懸吊式磷脂雙層膜來排除基材的影響，以利研究穿透式膜蛋白。懸吊式磷脂雙層膜是利用磷脂在介於有機相及水相的微孔洞中排列而形成類似三明治模型的磷脂雙層膜，排除了基材干擾。 本研究使用氣泡崩解沉降法製備數種磷脂膜，包括電中性及負電性磷脂膜。藉由調控氣泡大小(1.5 μl ~ 2.5 μl)改變成型的磷脂膜大小(0.6 mm ~ 1.2 mm)，也由增加吸取磷脂量來增加膜的磷脂密度。其製備過程易受震動影響，所以要盡量避免桌面震動。另外也成功觀察到POPC懸吊式磷脂雙層膜的形成。藉由調控微孔洞大小(0.1 mm ~ 0.7 mm)改變成型磷脂模大小(0.1 mm ~ 0.7 mm)。磷脂膜容易因上下壓力差過大而破裂，所以製備懸吊式磷脂雙層膜要注意磷脂膜上下壓力是否相近。 在以氣泡崩解沉降法製備好的磷脂膜加入酵素觀察其與磷脂膜的作用情形。觀察到膜水解酵素phospholipase A2與流動性POPC膜作用較凝膠態DPPC膜慢。E. coli細胞壁合成酵素之一undecaprenyl pyrophosphate synthase (UPPs)在不同基質下與E. coli細胞膜的吸附能力不同，並觀察到其吸附並非完全隨機，會從點而面擴展。
並列摘要
A supported lipid bilayer (SLB) is a simplified, planar, and artificial membrane on a solid substrate. It is very useful for the study of protein-membrane interactions. The vesicle fusion method is the most convenient way to prepare SLB. However, SLB prepared by the vesicle fusion has three disadvantages. First, the area of the membrane is too large for the study using fluorescence recovery after photobleaching on fluid membrane. Second, the thickness of the water layer between the membrane and the substrate is only about one nanometer, too thin to study transmembrane protein. Third, it is difficult to prepare a negatively charged lipid bilayer on a glass substrate, whose surface is also negatively charged. A fluid and micron-sized lipid bilayer is desirable for the observation of protein-membrane interactions by microscope. We had used knife stripping and air bubble stripping to create small snd isolated membrane, but these two methods caused irreproducible destructions on the edges of lipid bilayers. We therefore set up the method of bubble collapse deposition (BCD) to prepare isolated lipid bilayers in small sizes. The method involves three steps: First, an air bubble is touched to a lipid bilayer and then is covered by a monolayer of phospholipid. Second, the phospholipid covered air bubble is moved underwater to another clean glass plate. Third, the air is slowly withdrawn from the air bubble and a lipid bilayer is formed by bubble collapse deposition. We also developed a method to create suspended lipid bilayers for the study of transmembrane protein. A suspended lipid bilayer was prepared on a microhole between the interface of organic solvent and water. We used bubble collapse deposition to produce several isolated bilayers of various phospholipids, such as 1-palmitoyl-2-oleoyl-sn- glycero-3- phosphocholine (POPC) and E. coli membrane lipids. We controlled the air bubble volume (1.5 μl ~ 2.5 μl) to create bilayers with different sizes (0.6 mm ~ 1.2 mm in diameter) and increased the density of the BCD bilayer by increasing the contact time and area of the air bubble with the lipid source bilayer. We found that vibration on the platform of the setup will lead to failed experiments. In addition, a suspended POPC lipid bilayer was successfully formed on the microhole in the interface of n-decane and water. The size of the microhole was changed from 0.1 mm ~ 0.7 mm in diameter. The pressure difference between the organic and water interface can easily destroy the suspended lipid bilayer. So the pressure balance on the two sides of the bilayer is important. For applications, we added enzymes to the prepared membrane to examine the interactions between the enzymes and the membrane. We observed that phospholipase A2 hydrolyzed the fluid POPC bilayer more slowly than the gel-state DPPC bilayer. E. coli undecaprenyl pyrophosphate synthase (UPPs), a water soluble enzyme, adsorb to the bilayer of E. coli membrane extract to different extent under the presence of different substrates. The adsorption did not happen in a totally random way but expanded from points to areas.