【摘要】： 跨膜屏障是非病毒轉基因載體在基因轉染中面臨的主要障礙。為了設計一種可有效跨越細胞膜屏障的轉基因載體,本文將胍基引入支化聚乙烯亞胺,制備了胍基化聚乙烯亞胺非病毒載體。瓊脂糖凝膠電泳,溴化乙錠(EB)置換實驗表明胍基化聚乙烯亞胺可有效復合DNA,但在一定程度上削弱了載體和DNA之間的相互作用。透射電鏡下,胍基化聚乙烯亞胺/DNA復合物呈球狀分散粒子,且當復合比大于臨界復合比時,其粒徑均小于150納米。Zeta電位實驗表明胍基化聚乙烯亞胺/DNA復合物表面帶正電荷,且胍基化后載體的Zeta電位有所降低。以熒光素酶為報告基因考察胍基化聚乙烯亞胺載體對非洲綠猴腎(COS-7)細胞的基因轉染,結果表明與未改性聚乙烯亞胺相比,胍基化聚乙烯亞胺有著更高的轉染效率和更低的細胞毒性。藥物抑制實驗揭示,凹陷蛋白介導的細胞內吞作用對胍基化聚乙烯亞胺載體的高效表達起關鍵作用。同時,利用反式環(huán)己二醇所具有的非特異性打開核孔的獨特作用,將反式環(huán)己二醇與胍基化載體體系相結合,結果顯示載體效率有了進一步的提高,預示其應用于非分裂細胞的基因轉染的可行性。鑒于這些特點,胍基化聚乙烯亞胺有望成為高效低毒的非病毒載體而應用于體內轉基因應用中。
[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.
【學位授予單位】：天津大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R346

【共引文獻】

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1 楊莉;治療型脂膜超聲微泡的制備方法學研究[D];第三軍醫(yī)大學;2006年

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本文編號：2475698