末端修饰的PAMAM非病毒基因治疗载体的研究

发布时间：2018-04-11 10:05

本文选题：非病毒基因治疗载体 + PAMAM　；参考：《天津大学》2006年硕士论文

【摘要】： 在基因治疗的过程中,基因载体有着极其重要的作用。对于常用的阳离子聚合物/质粒DNA非病毒基因治疗载体来讲,其与质粒DNA之间的相互作用包括静电,疏水等。而在基因传递的过程中,疏水作用究竟起一个什么样的作用是一个受到重视的问题。本文研究的主要内容有: 首先,本文参考文献,在液相中对PAMAM(polyamidoamine)进行了表面修饰,为了体现疏水性的作用,我们选择了带有苯环的苯丙氨酸(Phe)和色氨酸(Trp)作为表面修饰物。核磁共振检测证明了PAMAM表面连接了足够的氨基酸残基,生成了表面既有氨基(在水溶液中会带正电荷)又有大量疏水基的PAMAM氨基酸修饰物。采用了质粒提取试剂盒,色谱法和聚乙二醇法提取了得到实验所需的质粒,并利用电泳和紫外吸收来检测了质粒的质量和浓度,使之能够用于后续的实验。为了考查新聚合物和质粒形成的复合物的自聚过程,琼脂糖凝胶电泳,溴乙锭荧光发射,多角度激光散射和透射电镜从不同的方面显示了聚合物和质粒DNA之间的作用和质粒的缩合。聚合物对于动物细胞的毒性实验和转染效率实验反应了新聚合物的生物毒性和最终的转染效率。在低的浓度下,聚合物有相对较弱的毒性,在电荷比例为5的时候,修饰Phe和Trp的聚合物能够大大提高绿色荧光蛋白在非洲绿猿肾细胞中的表达效率。最终说明,含疏水基的氨基酸在PAMAM表面的引入,改变了PAMAM和质粒DNA之间的自聚过程,并最终提高了细胞转染的效率,说明了疏水作用在基因传递系统中的重要作用,为阳离子聚合物基因载体今后的发展提供了一个重要的参考
[Abstract]:Gene vectors play an important role in gene therapy.For the commonly used cationic polymer / plasmid DNA non-viral gene therapy vector, its interaction with plasmid DNA includes electrostatic, hydrophobic, and so on.In the process of gene transmission, what kind of hydrophobic effect is paid attention to.The main contents of this paper are as follows:Firstly, the surface modification of PamAmidoamine was carried out in liquid phase with reference. In order to show the hydrophobicity, the phenylalanine (Phe) and tryptophan (Trp) with benzene ring were selected as the surface modifiers.Nuclear magnetic resonance (NMR) results show that the surface of PAMAM has enough amino acid residues to form PAMAM amino acid modifiers with both amino acids (which are positively charged in aqueous solution) and hydrophobic groups.The plasmids were extracted by using plasmid extraction kit chromatography and polyethylene glycol method. The quality and concentration of plasmids were determined by electrophoresis and UV absorption so that the plasmids could be used in subsequent experiments.In order to investigate the self-polymerization process of the complexes formed by new polymers and plasmids, agarose gel electrophoresis, fluorescence emission of bromoethidium,Multi-angle laser scattering and transmission electron microscopy show the interaction between polymer and plasmid DNA and the condensation of plasmids.The toxicity test and transfection efficiency experiment of polymer to animal cells reflect the biological toxicity and the final transfection efficiency of the new polymer.At a low concentration, the polymer has relatively weak toxicity. When the charge ratio is 5, the polymer modified with Phe and Trp can greatly improve the expression efficiency of green fluorescent protein in the kidney cells of the African ape.Finally, the introduction of hydrophobic amino acids on the surface of PAMAM changed the self-polymerization process between PAMAM and plasmid DNA, and finally improved the efficiency of cell transfection, which indicated the important role of hydrophobic action in gene transmission system.It provides an important reference for the future development of cationic polymer gene vector.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

【引证文献】