新型阳离子高分子基因载体系统的构建与评价

发布时间：2018-06-19 23:39

本文选题：基因载体 + 转染效率　；参考：《武汉理工大学》2015年硕士论文

【摘要】：基因治疗已经开始用于肿瘤、癌症及各种遗传性疾病的治疗当中,临床效果较传统治疗显著,且应用前景广阔。而要实现高效的基因治疗,设计高效低毒的基因载体是其中一个关键的环节。基因载体包括病毒类载体、非病毒类载体,相对于病毒载体,非病毒载体尤其是阳离子非病毒载体具有低细胞毒性、低免疫性、易合成等特点,已成为目前的研究热点。壳聚糖作为天然的多糖,具有无毒,可降解,生物相容性好,且分子中含有大量的氨基,能与质粒DNA复合形成纳米粒,已有大量报道将其作为基因载体应用于基因治疗。但是壳聚糖水溶性差,与DNA复合紧密而难于在体内释放DNA,转染效率较低,临床应用受到限制。因此,寻找适合的途径得到新型阳离子壳聚糖衍生物来作为基因载体,提高它的转染效率,是研究者努力的方向。本论文实验第一部分利用迈克尔加成反应合成了一系列新型水溶性壳聚糖衍生物。用核磁共振氢谱对载体材料进行化学表征,以确定其结构。用凝胶电泳实验观察壳聚糖衍生物与质粒DNA的结合能力,用电子扫描电镜来观察粒子的形状大小,测定粒子粒径及电动电势,用MTT法检测复合体的细胞毒性,通过体外细胞培养来测定壳聚糖衍生物的转染效率。实验结果表明,与壳聚糖相比,新型壳聚糖衍生物具有较低的细胞毒性,较好的DNA复合能力,在293 T细胞和HeLa细胞中有更高的转染效率。基于二硫键对不同浓度的谷胱甘肽浓度有特异响应性,并且细胞中的谷胱甘肽浓度高于血液中的谷胱甘肽浓度,癌细胞中的谷胱甘肽浓度约为正常细胞内的四倍,本论文实验第二部分我们以透明质酸为骨架,引入二硫键,后接枝四乙烯五胺制备出一种基于透明质酸的阳离子高分子载体,用红外、核磁共振氢谱(1H NMR)对载体材料进行结构表征。通过与质粒DNA形成复合物,并用琼脂糖凝胶电泳分析其结合DNA的能力及在高浓度谷胱甘肽刺激下释放DNA的能力;测定复合粒子的粒径、电势,并在扫描电子显微镜下观察复合物的形态;用MTT法检测复合体的细胞毒性。结果表明,新合成的透明质酸阳离子衍生物易与DNA复合,并在模拟细胞内高浓度谷胱甘肽刺激下释放DNA,且细胞毒性较低,是一种潜在的基因载体。设计开发新型的阳离子基因载体具有重要的意义。本论文通过对壳聚糖和透明质酸的化学修饰,分别得到相应的阳离子高分子衍生物,实验结果显示,所制备的新型阳离子高分子都具有较低的细胞毒性,较好的DNA复合能力,在作为基因载体方面具有潜在的应用。
[Abstract]:Gene therapy has been used in the treatment of cancer, cancer and various hereditary diseases. To achieve efficient gene therapy, the design of efficient and low-toxic gene vector is one of the key links. Gene vectors include viral vectors, non-viral vectors. Compared with viral vectors, non-viral vectors, especially cationic non-viral vectors, have the characteristics of low cytotoxicity, low immunity and easy synthesis. Chitosan, as a natural polysaccharide, is nontoxic, biodegradable, biocompatible, and has a large amount of amino acids in the molecule, which can compound with plasmid DNA to form nanoparticles. It has been reported that chitosan can be used as gene vector in gene therapy. However, chitosan is difficult to release DNA in vivo because of its poor water solubility and compounding with DNA, so its transfection efficiency is low, and its clinical application is limited. Therefore, to find a suitable way to obtain new cationic chitosan derivatives as gene vector and improve its transfection efficiency is the direction of researchers' efforts. In the first part of this thesis, a series of new water-soluble chitosan derivatives were synthesized by Michael addition reaction. The support material was chemically characterized by nuclear magnetic resonance spectroscopy (NMR) to determine its structure. The binding ability of chitosan derivatives to plasmid DNA was observed by gel electrophoresis, the shape and size of particles, the particle size and electromotive force were measured by electron scanning electron microscope (SEM), and the cytotoxicity of the complex was detected by MTT method. The transfection efficiency of chitosan derivatives was determined by cell culture in vitro. The results showed that compared with chitosan, the new chitosan derivatives had lower cytotoxicity, better DNA recombination ability and higher transfection efficiency in 293T cells and HeLa cells. Based on the specific response of disulfide bond to different concentrations of glutathione, the concentration of glutathione in cells was higher than that in blood, and the concentration of glutathione in cancer cells was about four times as high as that in normal cells. In the second part of this thesis, we used hyaluronic acid as the skeleton, introduced disulfide bond, and then grafted tetraethylenepentylamine to prepare a kind of cationic polymer carrier based on hyaluronic acid. The structure of the carrier was characterized by 1H NMR. By forming a complex with plasmid DNA, using agarose gel electrophoresis to analyze its ability to bind DNA and release DNA under high concentration of glutathione, to determine the particle size and potential, The morphology of the complex was observed under scanning electron microscope (SEM), and the cytotoxicity of the complex was detected by MTT assay. The results showed that the newly synthesized hyaluronic acid cationic derivatives were easy to compound with DNA and released DNA under the stimulation of high concentration of glutathione in mimic cells, and the cytotoxicity was low, so it was a potential gene vector. It is of great significance to design and develop a new cationic gene vector. In this paper, the corresponding cationic polymer derivatives were obtained by chemical modification of chitosan and hyaluronic acid. The experimental results showed that the new cationic polymers had lower cytotoxicity and better DNA recombination ability. It has potential application as gene vector.
【学位授予单位】：武汉理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450

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