脂质体制备和电融合实验研究

发布时间：2018-11-18 12:41

【摘要】：脂质体是磷脂双分子层膜在水相中组装成的内部为水相的闭合囊泡。脂质体具有双亲性、生物相容性，和细胞膜及细胞器膜具有类似的结构，在美容、食品、医药和生物化学研究等领域得到了广泛的应用。粒径大于1μm的大脂质体是一种常用的转染工具，另外它还是很好的探索细胞功能的工具，可以作为微反应器来模拟细胞或细胞器，研究体内的生物化学反应，以及细胞膜的结构、功能和膜穿孔、融合的机制等。大脂质体的制备方法主要有传统的薄膜蒸发法、两相蒸发法和新兴的电场制备法、微流控制备法等，各种方法制备得到的脂质体具有不同的特性。脂质体融合技术能够促发微量物质间的反应，模拟体内细胞或细胞器内的生物化学反应，实现在微观水平对生命体内的各种生物化学反应进行研究。另外在基因转染、细胞器监测、药物定点传送和细胞膜蛋白的生物物理学研究等方面有很重要的意义，还可作为研究细胞膜融合机理的模型。对脂质体融合技术的研究经历了物理促进融合、脂质体群电融合、微操作-电脉冲结合的融合法等几个发展阶段。其中微操作-电脉冲结合的脂质体融合法实现了对脂质体的可控融合，与其他方法相比具有精确操作、可控制性强、融合率高等优点，，但仍存在设备昂贵、操作方法复杂、耗时长等缺点。近年来，又涌现出一种基于微电极芯片的脂质体电融合技术。本课题组研制了多种电融合芯片，并成功应用于多种细胞的高效率电融合。脂质体融合和细胞融合的本质都是膜融合。为了制备一种适合于脂质体电融合的微电极芯片，本文将利用课题组已有的电融合芯片进行脂质体电融合实验，摸索脂质体融合的条件，为进一步开展脂质体-细胞融合和脂质体电融合芯片的研制奠定基础。本文首先对薄膜分散法、两相蒸发法、电场制备法等大脂质体制备方法进行了对比实验研究，同时研究了胆固醇含量以及离心处理对大脂质体稳定性的影响，并最终制备电融合用的脂质体悬液。然后利用课题组现有的两种电融合芯片分别搭建了实验平台，在平台上开展了脂质体电融合的相关实验：基于PI基底的电融合芯片和基于硅玻基底的电融合芯片上的脂质体排队实验研究；基于硅玻基底的电融合芯片上的脂质体电融合实验研究。在脂质体排队实验研究中，用基于硅玻基底的电融合芯片实现了脂质体排队，并且排队率较高。在脂质体电融合实验中，观察到了脂质体融合的现象。
[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.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R312

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