基于PGMA为骨架基因载体的构建及其性能研究

发布时间：2018-07-13 19:57

【摘要】：临床上所要求的成功基因载体不仅要有高的转染效率还要具有低的毒性。本课题主要以PGMA为骨架构建低毒高效的基因载体。具体实验内容如下： 1,利用乙醇胺(EA)与线性聚甲基丙烯酸缩水甘油酯(PGMA)进行开环反应,得到具有仲胺和非离子亲水性基团的阳离子聚合物PGEA。通过一系列的转染和毒性试验表明,PGEA在不同的细胞系中都表现出很高的细胞转染效率(高于25KD的聚乙烯亚胺(PEI)或与之相当)以及很低的细胞毒性(小于25KDPEI的10%)。 2,利用不同的胺类化合物对PGMA进行功能化(包括1-氨基-2-丙醇(AP1)功能化的PGAP1;3-氨基-2-丙醇(AP2)功能化的PGAP2；乙醇胺(EA)功能化的PGEA;乙醇胺/N,N-二甲基乙二胺(EA/DED)共同功能化的PGEADED;DED功能化的PGDED;季胺化的DED(QDED)功能化的QPGDED,比较它们作为基因载体的性能。与EA相比,AP1含有一个多余的甲基,而AP2含有一个多余的亚甲基,这使它们具有大致相同的缩合DNA能力,并能提高转染效率,从结果上看,PGAP1的转染效率是所有材料中最高的。DED含有叔胺基团,可以被进一步季胺化以增加表面正电荷,进而增强缩合DNA的能力,与PGDED目比,QPGDED的毒性没有明显增加,但DED以及季胺化的QDED会严重影响材料的缓冲能力,导致转染效率降低。本研究的主要目的是更好的构建PGMA系列的基因载体,使其具有优异的生物及物理性能。 3,进一步提高PGMA衍生物载体的可降解性和转染效率将有利于构建更好的基因传递体系。梳型阳离子聚合物作为非病毒基因载体已经受到关注和重视。我们利用ATRP和开环反应制备了具有可降解性的具有高分子量梳状的PGMA衍生物即(c-PGEA)载体。通过一系列的表征,结果证明梳状c-PGEA载体具有良好的络合DNA的能力、低细胞毒性和良好的缓冲能力。更重要的是,c-PGEA载体能提高基因的转染效率,并且它们具有降解性有利于PGEA最终从人体中排除。此外,c-PGEA的PGEA侧链还可以与一些聚(乙二醇)乙醚甲基丙烯酸酯(PEGEEMA)系列共聚以进一步提高基因转染效率。以上的结果都表明了PGEA系列作为阳离子基因载体已超过了金标PEI。因此,以PGMA为骨架的阳离子基因载体具有低的毒性、高的转染效率和可降解性会促进高效的基因载体体系的发展。有希望在未来的基因疗法中成为安全有效的基因载体。
[Abstract]:The successful gene vectors need not only high transfection efficiency but also low toxicity. In this study, PGMA was used as skeleton to construct low toxicity and high efficiency gene vector. The experimental contents are as follows: 1. The cationic polymer PGEAwith secondary amines and non-ionic hydrophilic groups was obtained by ring opening reaction of ethanolamine (EA) with linear polyglycidyl methacrylate (PGMA). A series of transfection and toxicity tests showed that PGEA showed high transfection efficiency (higher than or equivalent to PEI) and low cytotoxicity (less than 10% of 25KD PEI) in different cell lines. (2) PGMA was functionalized by different amines (including PGAP1, 3-amino-2-propanol (AP2) functionalized PGAP2; Ethanolamine (EA) functionalized PGEAA, ethanolamine / NN- dimethylethylenediamine (EA-DED) co-functionalized PGEADED functionalized PGDED, quaternary amine-DED (QDED) functionalized QPGDED, and their performance as gene vectors were compared. Compared with EA, AP1 contains an excess methyl and AP2 contains an excess methylene, which makes them have roughly the same condensation ability of DNA and can improve transfection efficiency. The results showed that the transfection efficiency of PGAP1 was the highest in all the materials, and it could be further quaternary amination to increase the surface positive charge and then enhance the ability of condensation DNA. Compared with PGDED, the toxicity of QPGDED was not significantly increased. But DED and quaternary amination QDED will seriously affect the buffer ability of the material, resulting in a decrease in transfection efficiency. The main purpose of this study is to construct the PGMA gene vector. 3. Further improving the degradability and transfection efficiency of PGMA derivative vector will be beneficial to the construction of a better gene transfer system. Comb-type cationic polymer as a non-viral gene vector has been paid attention to. Using ATRP and ring-opening reaction, the degradable PGMA derivative (c-PGEA) with high molecular weight comb shape was prepared. Through a series of characterization, it was proved that the comb c-PGEA carrier had good ability of complexing DNA, low cytotoxicity and good buffer ability. More importantly, c-PGEA vectors can improve the efficiency of gene transfection, and their degradability is conducive to the eventual exclusion of PGEA from human body. In addition, the PGEA side chain of c-PGEA can be copolymerized with some poly (ethylene glycol) ether methacrylate (PEGEEMA) series to further improve the efficiency of gene transfection. The above results indicate that the PGEA series as a cationic gene vector has exceeded the gold standard PEI. Therefore, the cationic gene vector based on PGMA has low toxicity, and high transfection efficiency and degradability will promote the development of efficient gene vector system. It is expected to become a safe and effective gene vector in gene therapy in the future.
【学位授予单位】：北京化工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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