胍基化聚乙烯亚胺非病毒转基因载体
发布时间：2019-05-13 07:19
【摘要】： 跨膜屏障是非病毒转基因载体在基因转染中面临的主要障碍。为了设计一种可有效跨越细胞膜屏障的转基因载体,本文将胍基引入支化聚乙烯亚胺,制备了胍基化聚乙烯亚胺非病毒载体。琼脂糖凝胶电泳,溴化乙锭(EB)置换实验表明胍基化聚乙烯亚胺可有效复合DNA,但在一定程度上削弱了载体和DNA之间的相互作用。透射电镜下,胍基化聚乙烯亚胺/DNA复合物呈球状分散粒子,且当复合比大于临界复合比时,其粒径均小于150纳米。Zeta电位实验表明胍基化聚乙烯亚胺/DNA复合物表面带正电荷,且胍基化后载体的Zeta电位有所降低。以荧光素酶为报告基因考察胍基化聚乙烯亚胺载体对非洲绿猴肾(COS-7)细胞的基因转染,结果表明与未改性聚乙烯亚胺相比,胍基化聚乙烯亚胺有着更高的转染效率和更低的细胞毒性。药物抑制实验揭示,凹陷蛋白介导的细胞内吞作用对胍基化聚乙烯亚胺载体的高效表达起关键作用。同时,利用反式环己二醇所具有的非特异性打开核孔的独特作用,将反式环己二醇与胍基化载体体系相结合,结果显示载体效率有了进一步的提高,预示其应用于非分裂细胞的基因转染的可行性。鉴于这些特点,胍基化聚乙烯亚胺有望成为高效低毒的非病毒载体而应用于体内转基因应用中。
[Abstract]:Transmembrane barrier is the main obstacle in gene transfection of non-viral transgenic vector. In order to design a transgenic vector which can effectively cross the cell membrane barrier, guanidine group was introduced into branched polyethylene imine to prepare guanidine polyleneimine non-viral vector. Agarose gel electrophoresis and ethidium bromide (EB) replacement test showed that guanidine polyleneimine could effectively compound DNA, but weakened the interaction between carrier and DNA to a certain extent. Under transmission electron microscope, guanidine polyleneimine / DNA complex is spherical dispersed particles, and when the composite ratio is greater than the critical composite ratio, The particle size of the composite was less than 150nm. Zeta potential test showed that there was a positive charge on the surface of guanidine polyleneimine / DNA complex, and the Zeta potential of the carrier decreased after guanidine conversion. Using luciferase as reporter gene to investigate the gene transfer of guanidine polyleneimine vector to African green monkey kidney (COS-7) cells, the results showed that compared with unmodified polyvinyleneimine, Guanidine polyvinylimine has higher transfection efficiency and lower cytotoxicity. The drug inhibition test showed that the endocytosis mediated by depression protein played a key role in the high expression of guanidine polyleneimine carrier. At the same time, taking advantage of the unique effect of trans-cyclohexanediol on opening nuclear pores, trans-cyclohexanediol was combined with guanidine carrier system, and the results showed that the carrier efficiency had been further improved. It indicates the feasibility of gene transfection in non-mitotic cells. In view of these characteristics, guanidine polyleneimine is expected to become a highly efficient and low toxic non-viral vector and used in transgenic applications in vivo.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346
本文编号：2475698
[Abstract]:Transmembrane barrier is the main obstacle in gene transfection of non-viral transgenic vector. In order to design a transgenic vector which can effectively cross the cell membrane barrier, guanidine group was introduced into branched polyethylene imine to prepare guanidine polyleneimine non-viral vector. Agarose gel electrophoresis and ethidium bromide (EB) replacement test showed that guanidine polyleneimine could effectively compound DNA, but weakened the interaction between carrier and DNA to a certain extent. Under transmission electron microscope, guanidine polyleneimine / DNA complex is spherical dispersed particles, and when the composite ratio is greater than the critical composite ratio, The particle size of the composite was less than 150nm. Zeta potential test showed that there was a positive charge on the surface of guanidine polyleneimine / DNA complex, and the Zeta potential of the carrier decreased after guanidine conversion. Using luciferase as reporter gene to investigate the gene transfer of guanidine polyleneimine vector to African green monkey kidney (COS-7) cells, the results showed that compared with unmodified polyvinyleneimine, Guanidine polyvinylimine has higher transfection efficiency and lower cytotoxicity. The drug inhibition test showed that the endocytosis mediated by depression protein played a key role in the high expression of guanidine polyleneimine carrier. At the same time, taking advantage of the unique effect of trans-cyclohexanediol on opening nuclear pores, trans-cyclohexanediol was combined with guanidine carrier system, and the results showed that the carrier efficiency had been further improved. It indicates the feasibility of gene transfection in non-mitotic cells. In view of these characteristics, guanidine polyleneimine is expected to become a highly efficient and low toxic non-viral vector and used in transgenic applications in vivo.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346
【共引文献】
相关硕士学位论文 前3条
1 杨莉;治疗型脂膜超声微泡的制备方法学研究[D];第三军医大学;2006年
2 章春花;鼠ING4基因腺病毒表达载体的构建及抗肿瘤效应的实验研究[D];苏州大学;2006年
3 刘国通;超声微泡介导转染FKBP12.6基因对小鼠H9c2（2-1）心肌细胞结构和功能的影响[D];第三军医大学;2007年
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