基于凝胶多糖的兼性高分子的合成及其核酸递送活性的研究

发布时间：2018-01-06 18:42

本文关键词：基于凝胶多糖的兼性高分子的合成及其核酸递送活性的研究　出处：《内蒙古大学》2017年硕士论文　论文类型：学位论文

【摘要】：RNA干扰(RNAi)是由双链RNA引起的一种高效的转录后水平基因沉默,在多种疾病的临床治疗上有巨大的应用潜力,其临床应用的关键之一是构建安全有效的核酸递送载体。本文以凝胶多糖(Curdlan)为原料,首先对其结构单元的C5位进行氧化及酸化,制备完全氧化的β-1,3-polyglucuronic acid,随后与四乙烯五胺(TEPA)通过酰胺反应制备凝胶多糖兼性高分子TEPAC-m(m=1,-2,-3)。经核磁共振波谱确定结构,通过激光粒度仪测定其粒径分布、Zeta电动电势,酸碱滴定法测定其对H+的缓冲能力。随后通过MTT法细胞毒性实验、体外细胞质粒和siRNA转染试验对其作为核酸载体的安全性及递送效率等做了研究。研究结果表明,此新型的兼性高分子具有良好的水溶性和质子海绵效应;对于A549和HeLa细胞株显示了低于6AC-100和PEI的细胞毒性;对于HeLa、HepG2和293T细胞株能够有效地递送质粒进入细胞内;对于HeLa和HepG2细胞株能较高效率地递送siRNA使内源靶mRNA表达水平显著下调。可见,此新型基于凝胶多糖的兼性高分子作为核酸载体具有潜在的进一步研发的价值。综合而言,本论文成功合成了水溶性凝胶多糖兼性高分子TEPAC-m(m=1,-2,-3),应用于核酸的体外递送,并较系统地研究了其作为核酸载体的安全性和递送效率。所得研究结果不但为RNAi提供了一种新型兼性高分子多糖载体,而且为后续核酸载体研发提供了基础与研究依据。
[Abstract]:RNA interference RNAi is a highly efficient post-transcriptional gene silencing induced by double-stranded RNA. It has great potential in clinical treatment of many diseases. One of the key clinical applications is to construct a safe and effective nucleic acid delivery vector. In this paper, the C5 site of its structural unit was oxidized and acidified with Curdlan-gel as the raw material. The completely oxidized 尾 -1C 3-polyglucuronic acid was prepared. Subsequently, a facultative polymer, TEPAC-mGMN, was prepared by reaction with tetraethylenepentylamine (TEPA). The structure was determined by nuclear magnetic resonance spectroscopy (NMR). The particle size distribution of Zeta electromotive force was measured by laser particle size analyzer, the buffer capacity to H was measured by acid-base titration, and the cytotoxicity test was carried out by MTT method. The cell plasmid and siRNA transfection test in vitro were used to study the safety and delivery efficiency of nucleic acid vector. The new facultative polymer has good water-solubility and proton sponge effect. The cytotoxicity of A549 and HeLa cells was lower than that of 6AC-100 and PEI. For HeLaG-2 and 293T cell lines, the plasmids were effectively delivered into the cells. For HeLa and HepG2 cell lines, the expression of endogenous target mRNA was significantly down-regulated by delivering siRNA efficiently. This new type of facultative polymer based on gel polysaccharides as a carrier of nucleic acid has potential value for further development. In this paper, a facultative polymer, TEPAC-mGN, was successfully synthesized, which was used to deliver nucleic acid in vitro. The safety and delivery efficiency of RNAi as nucleic acid carrier were systematically studied. The results not only provided a new facultative polymer polysaccharide carrier for RNAi. It also provides the basis and research basis for the research and development of nucleic acid carriers.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;O631

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