【摘要】：脂質(zhì)體是磷脂雙分子層膜在水相中組裝成的內(nèi)部為水相的閉合囊泡。脂質(zhì)體具有雙親性、生物相容性，和細胞膜及細胞器膜具有類似的結(jié)構(gòu)，在美容、食品、醫(yī)藥和生物化學(xué)研究等領(lǐng)域得到了廣泛的應(yīng)用。粒徑大于1μm的大脂質(zhì)體是一種常用的轉(zhuǎn)染工具，另外它還是很好的探索細胞功能的工具，可以作為微反應(yīng)器來模擬細胞或細胞器，研究體內(nèi)的生物化學(xué)反應(yīng)，以及細胞膜的結(jié)構(gòu)、功能和膜穿孔、融合的機制等。大脂質(zhì)體的制備方法主要有傳統(tǒng)的薄膜蒸發(fā)法、兩相蒸發(fā)法和新興的電場制備法、微流控制備法等，各種方法制備得到的脂質(zhì)體具有不同的特性。 脂質(zhì)體融合技術(shù)能夠促發(fā)微量物質(zhì)間的反應(yīng)，模擬體內(nèi)細胞或細胞器內(nèi)的生物化學(xué)反應(yīng)，實現(xiàn)在微觀水平對生命體內(nèi)的各種生物化學(xué)反應(yīng)進行研究。另外在基因轉(zhuǎn)染、細胞器監(jiān)測、藥物定點傳送和細胞膜蛋白的生物物理學(xué)研究等方面有很重要的意義，還可作為研究細胞膜融合機理的模型。對脂質(zhì)體融合技術(shù)的研究經(jīng)歷了物理促進融合、脂質(zhì)體群電融合、微操作-電脈沖結(jié)合的融合法等幾個發(fā)展階段。其中微操作-電脈沖結(jié)合的脂質(zhì)體融合法實現(xiàn)了對脂質(zhì)體的可控融合，與其他方法相比具有精確操作、可控制性強、融合率高等優(yōu)點，，但仍存在設(shè)備昂貴、操作方法復(fù)雜、耗時長等缺點。近年來，又涌現(xiàn)出一種基于微電極芯片的脂質(zhì)體電融合技術(shù)。本課題組研制了多種電融合芯片，并成功應(yīng)用于多種細胞的高效率電融合。脂質(zhì)體融合和細胞融合的本質(zhì)都是膜融合。為了制備一種適合于脂質(zhì)體電融合的微電極芯片，本文將利用課題組已有的電融合芯片進行脂質(zhì)體電融合實驗，摸索脂質(zhì)體融合的條件，為進一步開展脂質(zhì)體-細胞融合和脂質(zhì)體電融合芯片的研制奠定基礎(chǔ)。 本文首先對薄膜分散法、兩相蒸發(fā)法、電場制備法等大脂質(zhì)體制備方法進行了對比實驗研究，同時研究了膽固醇含量以及離心處理對大脂質(zhì)體穩(wěn)定性的影響，并最終制備電融合用的脂質(zhì)體懸液。然后利用課題組現(xiàn)有的兩種電融合芯片分別搭建了實驗平臺，在平臺上開展了脂質(zhì)體電融合的相關(guān)實驗：基于PI基底的電融合芯片和基于硅�；椎碾娙诤闲酒系闹|(zhì)體排隊實驗研究；基于硅�；椎碾娙诤闲酒系闹|(zhì)體電融合實驗研究。 在脂質(zhì)體排隊實驗研究中，用基于硅�；椎碾娙诤闲酒瑢崿F(xiàn)了脂質(zhì)體排隊，并且排隊率較高。在脂質(zhì)體電融合實驗中，觀察到了脂質(zhì)體融合的現(xiàn)象。
[Abstract]:Liposomes are closed vesicles of phospholipid bilayers assembled in aqueous phase. Liposomes have been widely used in many fields such as beauty food medicine and biochemistry because of their amphiphilic biocompatibility similar structure to cell membrane and organelle membrane. Large liposomes larger than 1 渭 m in diameter are commonly used as transfection tools. In addition, they can be used as microreactors to simulate cells or organelles, and to study biochemical reactions in vivo. And cell membrane structure, function and membrane perforation, fusion mechanism and so on. The preparation methods of large liposomes mainly include the traditional thin-film evaporation method, two-phase evaporation method and new electric field preparation method, microfluidic preparation method, etc. The liposomes prepared by various methods have different characteristics. Liposome fusion technology can stimulate the reaction between trace substances, simulate the biochemical reactions in cells or organelles in vivo, and realize the study of various biochemical reactions in vivo at the microcosmic level. In addition, it is of great significance in gene transfection, organelle monitoring, drug delivery and biophysical study of cell membrane proteins. It can also be used as a model to study the mechanism of cell membrane fusion. The research on liposome fusion has gone through several stages, such as physical fusion, electrofusion of liposome, micromanipulation and electric pulse fusion. The liposome fusion method with micromanipulation and electric pulse binding can realize the controllable fusion of liposome. Compared with other methods, it has the advantages of precise operation, strong controllability and high fusion rate, but the equipment is expensive and the operation method is complex. Time-consuming and other shortcomings. In recent years, a liposome electrofusion technology based on microelectrode chip has emerged. A variety of electrofusion chips have been developed and successfully applied to the high efficiency electrofusion of many cells. The essence of liposome fusion and cell fusion is membrane fusion. In order to prepare a kind of microelectrode chip suitable for liposome electrofusion, the experiment of liposome electrofusion was carried out by using the existing electrofusion chip, and the conditions of liposome fusion were explored. It lays a foundation for the further development of liposome-cell fusion and liposome electrofusion chip. In this paper, the preparation methods of large liposomes, such as thin-film dispersion, two-phase evaporation and electric field preparation, were studied, and the effects of cholesterol content and centrifugation on the stability of large liposomes were studied. Finally, the liposome suspension for electrofusion was prepared. Then the experiment platform is built by using two kinds of electric fusion chips. The experiments of liposome electrofusion were carried out on the platform: the electrofusion chip based on the PI substrate and the liposome queuing experiment on the electrofusion chip based on the silica glass substrate; Experimental study of Liposome Electrofusion on Silicon-glass substrate Electrofusion Chip. In the study of liposome queuing, the electrofusion chip based on silicon glass substrate was used to realize the liposome queuing, and the queuing rate was high. In the experiment of liposome electrofusion, the phenomenon of liposome fusion was observed.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R312

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