细胞表面工程在细胞膜成像中的应用

发布时间：2018-04-12 21:06

本文选题：细胞表面工程 + 细胞膜成像　；参考：《东南大学》2016年博士论文

【摘要】：细胞膜是一种关键细胞器,它在结构上构成了细胞的边界以保持整个细胞的完整性,又在功能上与细胞贴壁、迁移、增殖、胞吞、凋亡和信号转导等重要生命活动密切相关。对细胞膜结构与功能的研究可以获得关于细胞行为和状态的重要信息。细胞膜荧光标记是细胞膜研究中最简单和最重要的手段,它不仅可以直观地观察细胞膜的结构,还可以动态追踪其变化。目前常用的细胞膜荧光染料(如DiD家族和FM家族)面临着容易被细胞内吞、荧光稳定性不足以及与免疫荧光染色不兼容等诸多问题。另一方面,近年来兴起的细胞表面工程技术,可以通过修饰细胞表面从而赋予细胞新的性质和功能,因此引起越来越多的关注。本论文采用以疏水锚定为修饰策略的细胞表面工程技术,设计合成了若干新型细胞膜荧光成像试剂,大大改善了细胞膜的成像效果。由于胆固醇分子具有优异的细胞膜疏水锚定能力,我们首先基于胆固醇分子开发出了带生物素基团的单位点细胞膜锚定试剂(cholesterol-polyethylene glycol-biotin,chol-PEG-biotin)。通过该单位点膜锚定试剂使细胞表面生物素化后,可以高效地把修饰有亲和素分子的量子点聚集到细胞膜上,从而实现基于量子点的抗光漂白细胞膜标记,使长时间细胞膜荧光成像观察成为了可能。为了抑制细胞对细胞膜标记试剂的内吞作用,我们又进一步开发了多位点膜锚定试剂。该试剂以乙二醇壳聚糖(GC)高分子为骨架,侧链以末端接有胆固醇基团的聚乙二醇链段(PEG-cholesterol)为多位点锚定单元,以异硫氰酸荧光素(FITC)为荧光单元。由于该试剂具有多个膜锚定位点,而且分子量大,所以其被细胞内吞的程度大大减弱。除此之外,我们将多位点锚定策略升级为由生物素与亲和素介导的双重修饰法,避免了多位点试剂容易从细胞膜脱落而使细胞膜上荧光信号减弱的缺陷。该方法不仅将成像保持至8 h以上,而且实现了免清洗细胞膜成像。另外,我们还发现多位点锚定分子具有构象调整能力,使其不仅能够通过疏水锚定作用实现动物细胞膜成像,还可通过静电相互作用实现真菌和细菌的细胞壁成像,使细胞表面标记具有普适性。此外,免疫荧光染色时,由于多位点试剂带有大量的氨基基团,可使其在多聚甲醛固定细胞的过程中与膜蛋白间发生交联,从而能耐受表面活性剂的透化处理。这种抗透化特性使其能够兼容免疫荧光染色,实现细胞膜与细胞胞内蛋白的同时成像。总之,本论文所开发的各种细胞膜荧光标记策略不仅在功能上提高了细胞膜成像的效果,而且也有助于深入理解生物材料与细胞的相互作用机制,进而推动细胞表面工程技术的发展。
[Abstract]:Cell membrane is a key organelle, which structurally forms the boundary of cell to maintain the integrity of the whole cell, and is closely related to cell adhesion, migration, proliferation, cytoplasm, apoptosis and signal transduction.Important information about cell behavior and state can be obtained by studying the structure and function of cell membrane.Fluorescent labeling of cell membrane is the simplest and most important method in the study of cell membrane. It can not only observe the structure of cell membrane intuitively, but also dynamically track its changes.At present, common fluorescent dyes (such as DiD family and FM family) face many problems, such as easy to be swallowed by cells, lack of fluorescence stability and incompatible with immunofluorescence staining.On the other hand, the cell surface engineering technology developed in recent years can endow the cell with new properties and functions by modifying the cell surface, so it has attracted more and more attention.In this paper, several novel fluorescent imaging reagents of cell membrane were designed and synthesized using hydrophobic anchoring as the modification strategy, which greatly improved the imaging effect of cell membrane.Because of the excellent membrane hydrophobic anchoring ability of cholesterol molecules, we first developed a unit cell membrane anchoring reagent, cholesterol-polyethylene glycol-biotininchol-PEG-biotininine, based on cholesterol molecules.After the biotinylation of the cell surface was made by the unit dot membrane anchoring reagent, the quantum dots modified with affinity molecules could be efficiently aggregated onto the cell membrane, thus realizing the anti-photobleaching cell membrane marker based on the quantum dots.It is possible to observe long-term fluorescence imaging of cell membrane.In order to inhibit the endocytosis of cell membrane labeling reagents, we further developed multisite membrane anchoring reagents.The reagents are composed of ethylene glycol chitosan (GC) macromolecule, side chain with PEG-cholesterol terminated as multi-site anchoring unit and fluorescein isothiocyanate (FITC) as fluorescence unit.Because the reagent has many membrane anchoring sites and its molecular weight is high, its degree of endocytosis is greatly weakened.In addition, we upgrade the multi-site anchoring strategy to a biotinylated and avidin mediated double modification method, which avoids the defect that the multisite reagents are easy to fall off the cell membrane and weaken the fluorescence signal on the cell membrane.This method not only keeps the imaging up to more than 8 h, but also realizes clean-free cell membrane imaging.In addition, we also found that multi-site anchoring molecules have the ability to adjust conformation so that they can be imaged not only by hydrophobic anchoring, but also by electrostatic interaction between fungi and bacteria.Make cell surface marking universal.In addition, in immunofluorescence staining, the multi-site reagents have a large number of amino groups, which can make them cross link with membrane proteins during the process of formaldehyde fixation, so that they can withstand the permeation treatment of surfactants.This anti-permeability property enables it to be compatible with immunofluorescence staining and to achieve simultaneous imaging of cellular and cellular proteins.In conclusion, the various fluorescent labeling strategies developed in this paper not only improve the function of cell membrane imaging, but also contribute to understanding the interaction mechanism between biomaterials and cells.And then promote the development of cell surface engineering technology.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.0

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