侧链可还原降解的聚乙烯亚胺衍生物的合成和体外基因释放

发布时间：2018-01-09 08:08

  本文关键词：侧链可还原降解的聚乙烯亚胺衍生物的合成和体外基因释放　出处：《苏州大学》2010年硕士论文　论文类型：学位论文

  更多相关文章： 基因转染 聚乙烯亚胺 复合物 质粒DNA 生物降解 双硫键

【摘要】：基因治疗就是将外源基因引入受感染的细胞内进行适当的表达,以纠正致病基因所产生的缺陷,从而达到从根本上治疗疾病的目的。 目前实施基因治疗的主要瓶颈是缺乏安全高效的基因传递系统。尽管病毒载体具有较高的转染效率,但存在制备复杂,有安全隐患,包装容量有限等问题。因此,近年来人们越来越青睐于人工合成的非病毒载体。由于阳离子聚合物作为非病毒性载体有安全性好、制备简单方便、可多次重复使用等众多优点而受到研究者们的青睐。其中,聚乙烯亚胺(PEI)因其正电荷密度较高,且可通过“质子海绵作用”逃离内涵体,以其结构为基础的新型阳离子聚合物载体成为研究热点。25 kDa支化PEI是迄今为止转染效率最高的阳离子聚合物载体之一,现已经被作为新开发的体外转染载体的比较标准,但其跟病毒性基因载体相比转染效率仍然较低且在体内不可降解,细胞毒性较大。 本论文研究的主要目的在于对转染效果较好的基因载体25 kDa支化PEI进行改性,引入在细胞内可还原降解的二硫键,制备出新型可降解的聚乙烯亚胺(PEI)衍生物作为基因载体,从而实现高效低毒的非病毒基因转染。25 kDa支化PEI首先分别与单体Ac-Cys-t~Boc及单体MAc-Cys-t~Boc进行迈克尔加成,然后脱保护形成取代度分别为14、27、34和13、19、38的PEI-Cys衍生物,并分别命名为PEI-(Cys)x(Ac)和PEI-(Cys)x(MAc)。随后对这些聚合物进行了一系列的性能表征。缓冲能力测试表明所有PEI-Cys衍生物的缓冲能力都比25 kDa支化PEI高(21.2-23.1%比15.1%);凝胶电泳结果表明胱胺的修饰有利于聚合物更好的压缩DNA,PEI-(Cys)x(Ac)在N/P≥5/1时可以将DNA压缩为80-90 nm的小粒子,比聚合物25 kDa支化PEI压缩的复合物粒径小(100-130 nm)。与之对比,聚合物PEI-(Cys)x(MAc)压缩DNA形成的复合物粒子则较大(100-180 nm,N/P从30/1到5/1);凝胶电泳及动态光散射(DLS)测试表明PEI-Cys衍生物与DNA形成的复合物在10 mM二硫苏糖醇(DTT)的溶液中可以快速地释放出质粒DNA;与25 kDa支化PEI相比所有的PEI-Cys衍生物都具有更低的细胞毒性(IC50: 100 mg/L versus ca. 11 mg/L);报告基因pGL3在HeLa细胞和293T细胞中的体外基因转染实验表明,随着取代度(DS)的提高,PEI-Cys衍生物的转染效率逐渐降低,而且在相似取代度的前提下聚合物PEI-(Cys)_x(MAc)比聚合物PEI-(Cys)_x(Ac)的转染效率高。在无血清和有血清条件下,聚合物PEI-(Cys)14(Ac)及PEI-(Cys)_x(MAc)与DNA在N/P比为10条件下形成的复合物的转染效率都明显高于25 kDa支化PEI(最高达4.1倍)。结果表明经胱胺修饰的可还原降解的新型PEI载体有较好的发展前景,可望被成功研制为既高效又安全无毒的新一代非病毒性基因转染试剂,应用于体内外基因转染。
[Abstract]:Gene therapy is to introduce foreign genes into infected cells for proper expression, in order to correct the defects of pathogenic genes, so as to fundamentally cure diseases. At present, the main bottleneck of gene therapy is the lack of a safe and efficient gene transmission system. Although the virus vector has a high transfection efficiency, but the preparation of complex, there are hidden dangers of safety. Packaging capacity is limited. Therefore, in recent years, people are more and more in favor of synthetic non-viral carriers. As cationic polymers as non-viral carriers have good security, simple and convenient preparation. Polyethyleneimide (PEI) is favored by researchers because of its high positive charge density, and it can escape from the connotation by "proton sponge action". The novel cationic polymer carrier based on its structure has become a hot topic. 25 kDa branched PEI is one of the most efficient cationic polymer carriers so far. Now it has been used as the comparative standard of the newly developed in vitro transfection vector, but its transfection efficiency is still lower than that of viral gene vector, which is not degradable in vivo and has greater cytotoxicity. The main purpose of this study was to modify the transfected gene vector 25 kDa branched PEI and introduce the reductive and degradable disulfide bonds in the cells. A novel biodegradable PEI derivative was prepared as a gene carrier. So as to achieve high efficiency and low toxicity of non-viral gene transfection. 25. KDa branched PEI was firstly added to monomer Ac-Cys-t~Boc and monomeric MAc-Cys-t~Boc respectively. Then the PEI-Cys derivatives with the degree of substitution of 14 ~ (27) ~ (34) and 13 ~ (19) ~ (38) were formed by deprotection. They were named PEI-CysCysX Acand (PEI-CysCysX) and PEI-CysCysTX (MAcl), respectively. The buffering ability of all PEI-Cys derivatives was higher than that of 25 kDa branched PEI. 21.2-23.1% vs 15.1%; The results of gel electrophoresis showed that the modification of cysteamine was beneficial to the better compression of DNA. DNA can be compressed to 80-90 nm particles at N / P 鈮，

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