本文選題：細(xì)胞表面工程 + 細(xì)胞膜成像��； 參考：《東南大學(xué)》2016年博士論文

【摘要】：細(xì)胞膜是一種關(guān)鍵細(xì)胞器,它在結(jié)構(gòu)上構(gòu)成了細(xì)胞的邊界以保持整個(gè)細(xì)胞的完整性,又在功能上與細(xì)胞貼壁、遷移、增殖、胞吞、凋亡和信號(hào)轉(zhuǎn)導(dǎo)等重要生命活動(dòng)密切相關(guān)。對(duì)細(xì)胞膜結(jié)構(gòu)與功能的研究可以獲得關(guān)于細(xì)胞行為和狀態(tài)的重要信息。細(xì)胞膜熒光標(biāo)記是細(xì)胞膜研究中最簡(jiǎn)單和最重要的手段,它不僅可以直觀地觀察細(xì)胞膜的結(jié)構(gòu),還可以動(dòng)態(tài)追蹤其變化。目前常用的細(xì)胞膜熒光染料(如DiD家族和FM家族)面臨著容易被細(xì)胞內(nèi)吞、熒光穩(wěn)定性不足以及與免疫熒光染色不兼容等諸多問題。另一方面,近年來興起的細(xì)胞表面工程技術(shù),可以通過修飾細(xì)胞表面從而賦予細(xì)胞新的性質(zhì)和功能,因此引起越來越多的關(guān)注。本論文采用以疏水錨定為修飾策略的細(xì)胞表面工程技術(shù),設(shè)計(jì)合成了若干新型細(xì)胞膜熒光成像試劑,大大改善了細(xì)胞膜的成像效果。由于膽固醇分子具有優(yōu)異的細(xì)胞膜疏水錨定能力,我們首先基于膽固醇分子開發(fā)出了帶生物素基團(tuán)的單位點(diǎn)細(xì)胞膜錨定試劑(cholesterol-polyethylene glycol-biotin,chol-PEG-biotin)。通過該單位點(diǎn)膜錨定試劑使細(xì)胞表面生物素化后,可以高效地把修飾有親和素分子的量子點(diǎn)聚集到細(xì)胞膜上,從而實(shí)現(xiàn)基于量子點(diǎn)的抗光漂白細(xì)胞膜標(biāo)記,使長時(shí)間細(xì)胞膜熒光成像觀察成為了可能。為了抑制細(xì)胞對(duì)細(xì)胞膜標(biāo)記試劑的內(nèi)吞作用,我們又進(jìn)一步開發(fā)了多位點(diǎn)膜錨定試劑。該試劑以乙二醇?xì)ぞ厶?GC)高分子為骨架,側(cè)鏈以末端接有膽固醇基團(tuán)的聚乙二醇鏈段(PEG-cholesterol)為多位點(diǎn)錨定單元,以異硫氰酸熒光素(FITC)為熒光單元。由于該試劑具有多個(gè)膜錨定位點(diǎn),而且分子量大,所以其被細(xì)胞內(nèi)吞的程度大大減弱。除此之外,我們將多位點(diǎn)錨定策略升級(jí)為由生物素與親和素介導(dǎo)的雙重修飾法,避免了多位點(diǎn)試劑容易從細(xì)胞膜脫落而使細(xì)胞膜上熒光信號(hào)減弱的缺陷。該方法不僅將成像保持至8 h以上,而且實(shí)現(xiàn)了免清洗細(xì)胞膜成像。另外,我們還發(fā)現(xiàn)多位點(diǎn)錨定分子具有構(gòu)象調(diào)整能力,使其不僅能夠通過疏水錨定作用實(shí)現(xiàn)動(dòng)物細(xì)胞膜成像,還可通過靜電相互作用實(shí)現(xiàn)真菌和細(xì)菌的細(xì)胞壁成像,使細(xì)胞表面標(biāo)記具有普適性。此外,免疫熒光染色時(shí),由于多位點(diǎn)試劑帶有大量的氨基基團(tuán),可使其在多聚甲醛固定細(xì)胞的過程中與膜蛋白間發(fā)生交聯(lián),從而能耐受表面活性劑的透化處理。這種抗透化特性使其能夠兼容免疫熒光染色,實(shí)現(xiàn)細(xì)胞膜與細(xì)胞胞內(nèi)蛋白的同時(shí)成像�？傊�,本論文所開發(fā)的各種細(xì)胞膜熒光標(biāo)記策略不僅在功能上提高了細(xì)胞膜成像的效果,而且也有助于深入理解生物材料與細(xì)胞的相互作用機(jī)制,進(jìn)而推動(dòng)細(xì)胞表面工程技術(shù)的發(fā)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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R318.0

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