【作者】 王峰；

【导师】 卢晓林；

【作者基本信息】 东南大学 ， 生物医学工程， 2020， 硕士

【摘要】 本论文通过构建DPPC生物仿生膜,并利用和频振动光谱(SFG)、共聚焦荧光显微镜(LSCM)等多种表征手段,在分子尺度下系统研究了磷脂酶PLA1与DPPC磷脂膜的水解反应机理,在该水解反应进行的过程当中,界面上由于不同分子之间的相互作用力,这些分子在界面上呈现出不同的取向,从而形成了一些特定的结构域。脂质代谢是细胞重要的生命活动组成部分之一,本实验研究为脂质代谢中相关的界面生化过程提供了重要的参考信息。具体为,构建了两种不同的细胞膜模型:PS(聚苯乙烯)-dDPPC磷脂单分子层、dDPPC-DPPC磷脂双分子层,研究了磷脂分子DPPC在磷脂酶PLA1作用下的水解过程。利用SFG的界面选择性,通过分析界面上不同分子所产生的SFG信号在水解过程中发生的变化,可以在分子尺度上揭示这一水解反应过程中不同分子的行为。研究过程中还采用了荧光共定位成像的方法,通过LSCM观测,追踪水解反应过程中不同分子取向的动态过程以及整个磷脂膜的形态学变化过程。实验结果表明,磷脂酶PLA1在水解DPPC磷脂分子过程中,会剪切甘油骨架上的sn-1酯键,水解产物之一的溶血磷脂大部分从界面上脱离并游离到溶液介质当中,而另一种水解产物脂肪酸则在界面上与磷脂酶PLA1自组装形成一种稳定的结构堆积在界面上,这种由脂肪酸构成的疏水区域被称为PLA1诱导结构域。本研究以界面磷脂水解反应为例,建立了一个基于现代光学技术、能够在分子和微观层面上表征界面动力学过程信息的实验体系,对于探究和理解磷脂水解的这一重要的生物物理化学过程提供了重要的实验数据与模型依据。进一步的,PLA1诱导结构域在界面上主要呈现出较强的疏水性,由于大多数生物蛋白质都具有两亲性,界面上的疏水相互作用,使得这些蛋白质会被非特异吸附在PLA1诱导结构域。这一现象与人体心血管疾病的发生有一定的联系,对这一类疾病的致病机理有着潜在的参考价值。更多还原

【Abstract】 In this thesis,the hydrolysis mechanism of the phospholipid（DPPC）biomimetic membrane catalyzed by phospholipase A1 was comprehensively studied via several characterization methods such as sum frequency vibration spectroscopy（SFG）method and confocal fluorescence microscopy（LSCM）method at the molecular scale.During this hydrolysis reaction,different molecules showed their own behavior at the interface due to the interaction force,and some specific domains were formed.This experimental study provides the essential information on revealing the interfacial biochemical process related to the metabolism of the lipids,which is one of the basic building blocks for cells.The interfacial hydrolysis of phospholipids catalyzed by phospholipase A₁（PLA₁）was studied via sum frequency generation（SFG）vibrational spectroscopy and fluorescence microscopy.Both monolayer（PS-dDPPC）and bilayer（dDPPC-DPPC）setups were used to confirm the hydrolysis mechanism.With the help of SFG method,the behavior of different molecules during this hydrolysis reaction was revealed by analyzing the changes of SFG signals from different molecules.Fluorescence co-localization imaging was also used in this research.Through the LSCM method,the dynamic process of different molecular behavior during the hydrolysis reaction and the morphological changes of the entire phospholipid membrane were tracked.The experimental results showed that phospholipase A₁ cleaved the sn-1 ester bond on the glycerol backbone.During the hydrolysis,lysophospholipids,one of the hydrolysis products,were desorbed from the interface into the solution,while the other products,fatty acids,self-organized and accumulated with PLA₁ at the interface to form the PLA₁ induced regions.In this study,an interfacial phospholipid hydrolysis reaction is used as an example to establish an experimental system based on modern optical technology which can characterize the information of interfacial molecules and it provided important experimental data and theoretical basis for exploring and understanding this important biophysical and chemical process of phospholipid hydrolysis.In addition,the hydrophobic PLA₁ induced region can serve as nonspecific binding domains for proteins and may lead to understanding of human vascular diseases from the perspective of this study.更多还原