葡聚糖—精胺阳离子聚合物基因载体体外基因转染的研究

发布时间：2018-02-05 17:42

本文关键词： 葡聚糖-精胺阳离子聚合物转染率复合物基因枪子弹　出处：《南方医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 背景：基因治疗是通过插入治疗基因至病变细胞内而产生特定功能的蛋白质或抑制异常基因表达，从而治疗疾病的一种新方法。基因治疗中首要难题是缺乏高效、安全及靶向的基因导入系统，即能将治疗基因输送至特定的靶细胞内并高效表达的、无毒或低毒的基因载体。目前基因载体的主要分为两类：病毒载体或非病毒载体。虽然非病毒载体在体内外基因转染效率低于病毒载体，且基因表达时间短，但其具有低免疫原性、能携带大分子量DNA及生物安全性高等特点而倍受青睐。阳离子聚合物是非病毒载体中最有潜力的一类载体系统，它们可通过结构修饰改变其物理化学及生物学性质。目的：合成水溶性葡聚糖-精胺阳离子聚合物基因载体DSP，研究以DSP为粘合剂基因枪子弹的制备方法、DSP／DNA复合物和基因枪子弹的生物物理学性质、初步探讨复合物的理化性质对其转染能力的影响。方法：研究以平均分子量为40KDa的葡聚糖作为反应起始物，在室温下经高碘酸钾氧化6个小时，得到了氧化葡聚糖。通过盐酸羟胺法测定了氧化葡聚糖醛基的含量，根据此含量将一定摩尔比的精胺与氧化葡聚糖反应，该反应能生成含希夫氏碱的葡聚糖-精胺亚胺聚合物，所得到的亚胺聚合物在过量硼氢化钠作用下，可还原生成稳定葡聚糖—精胺共价阳离子聚合物(DSP)。DSP的总含氮量及伯胺的含量分别通过元素分析仪及TNBS法测定，并计算了所合成DSP的接枝度及支化度；FT-IR及~1H-NMR鉴定了DSP化学结构。采用激光粒度仪测定复合物溶液的粒径和Zeta电位；琼脂糖凝胶电泳法考察DSP与DNA的结合能力，及DSP对DNA在核酸酶Ⅰ作用下的保护能力；滴定法考察DSP的在酸性条件下的缓冲能力；复合物体外转染SMMC-7721肝癌细胞及BHK-21细胞，通过荧光倒置倒置显微镜观察复合物对两株细胞的转染情况并计算转染率；MTT法考察复合物细胞毒性。在此基础上，研究了以DSP为粘合剂制备的基因枪子弹，并通过琼脂糖凝胶电泳、核酸酶降解试验和MTT法，分别研究了子弹的稳定性，，抗核酸酶Ⅰ能力及细胞毒性。结果：所合成氧化葡聚糖醛基含量为8.47±0.15 mmol／g，DSP接枝度为38.9％，支化度为38.5％。DSP能通过其带正电荷的氨基基团能与DNA带负电荷的磷酸基团通过静电吸附而将DNA完全压缩并中和其负电荷，使DNA缩合形成复合物。复合物质量比(DSP／DNA)在4∶1至20∶1之间，能形成稳定的复合物；复合物粒径范围为162.6-187.9nm，Zeta电位则从+8.45mV增至+39.6mV；DSP能充分压缩DNA并与之紧密吸附；DSP能有效保护DNA不受核酸酶Ⅰ降解，同时在一定pH范围内载体具有较强的缓冲能力；复合物在质量比为8∶1时(此时粒径为170.8±23.9nm，Zeta电位为+18.3±1.55mV)，对SMMC-7721肝癌细胞、BHK-21细胞的转染率均达到最高，分别为27.0±4.42％及22.2±4.63％，其效果均与Lipofectamine 2000相当；复合物的毒性随质粒浓度及DSP／DNA质量比增加；所制备的基因枪子弹溶液在不同DSP／DNA．及DNA／金比例下均能粘合且能保护质粒不受核酸酶降解；金含量越高，DSP／DNA比例越大，细胞毒性则越大。结论：该研究表明DSP／DNA复合物的粒径、电位、稳定性、抗核酸酶能力、缓冲容量及细胞毒性对其转染能力具有一定影响。所合成的葡聚糖-精胺阳离子聚合物是一种制备工艺简单、高效、低毒的基因载体，有望今后用于临床基因治疗。以DSP制备的基因枪子弹抗核酸酶能力强，有望提高体内基因枪转染效率。
[Abstract]:Background: gene therapy is caused by the insertion of specific functional gene therapy to the lesion within the cell protein or inhibit abnormal gene expression, a new method to treat the disease. The primary problem in gene therapy is the lack of efficient gene delivery system to the safety and the target, which can be delivered to the target cells for gene therapy in particular and high expression of gene carrier, non-toxic or low toxicity. At present is mainly divided into two categories: gene viral vectors or non viral vectors. Although non viral vectors in vitro and in vivo gene transfection efficiency than viral vectors, and gene expression in a short time, but it has low immunogenicity, which can carry large molecular weight and DNA biological safety is high and popular. The cationic polymer is a kind of vector system has the most potential non viral vector, which can be changed by modification of the structure of its physical chemistry and biology. Quality.
Objective: to synthesize water-soluble dextran spermine cationic polymer gene carrier DSP, preparation methods of adhesive gene gun bullets with DSP, DSP / DNA complex biophysical properties and gene gun bullets, preliminary study of the effects of physicochemical properties of the complexes on the transfection efficiency.
Methods: To study the average molecular weight of 40KDa dextran as the starting reaction at room temperature by potassium periodate oxidation of 6 hours, the content of oxidized dextran. Oxidized dextran aldehyde was determined by hydroxylamine hydrochloride method, according to the content of the oxidized dextran spermine and reaction molar ratio, the reaction can be generated containing Schiff base dextran spermine imine polymer obtained, imine polymer in excess of sodium borohydride under the action of reducing the formation of stable covalent dextran spermine cationic polymer (DSP) content of total nitrogen and primary amine.DSP were determined by elemental analysis and TNBS, the grafting degree and support the degree of the synthesis of DSP and FT-IR calculation; and ~1H-NMR identification of DSP chemical structure determination of complex solution by laser particle size analyzer and the particle size and Zeta potential; agarose gel electrophoresis of DSP and DNA Combining ability, and DSP ability to protect DNA nuclease in 1 under the action of DSP was examined by titration; under the condition of acid buffering capacity; compound in vitro transfection of SMMC-7721 cells and BHK-21 cells, observe the complex on two cell transfection and transfection efficiency was calculated by inverted fluorescence microscope; MTT method effects of complex cell toxicity. On this basis, the research on DSP adhesive preparation of gene gun bullets, and by agarose gel electrophoresis, nuclease degradation test and MTT method, the stability of the bullet, nuclease resistant ability and cytotoxicity of 1.
Results: the content of synthetic oxidized dextran aldehyde is 8.47 + 0.15 mmol / g, the grafting degree of DSP is 38.9%, the phosphate group amino groups branching degree of 38.5%.DSP through its positively charged energy and negative charge with DNA through electrostatic adsorption and DNA completely compressed and neutralize the negative charge, the DNA condensation forming compound at complex mass ratio (DSP / DNA) between 4: 1 to 20: 1, complexes can form stable complexes; particle size in the range of 162.6-187.9nm, the Zeta potential is increased from +8.45mV to +39.6mV; DSP and DNA can be fully compressed tightly with adsorption; DSP can effectively protect DNA from nucleic acid I enzyme degradation, at the same time in a certain range of pH carrier has strong buffering capacity; complex in a mass ratio of 8: 1 (when the particle size is 170.8 + 23.9nm, the Zeta potential of +18.3 + 1.55mV), on SMMC-7721 hepatoma cells, the transfection rate of BHK-21 cells reached the highest point. Don't be 27 + 4.42% and 22.2 + 4.63%, and the results were equivalent to Lipofectamine 2000; the toxicity of compounds with plasmid concentration and DSP / DNA mass ratio increased; the solution preparation of gene gun bullets can adhesion and to protect DNA from nuclease degradation in different DSP / DNA. and DNA / Au ratios were; the higher the gold content and DSP / DNA ratio is greater, the greater the cytotoxicity.
Conclusion: the results showed that the DSP / DNA composite particle size, potential, stability, nuclease resistance, buffer capacity and cell toxicity has certain effect on the transfection ability. The synthesis of dextran spermine cationic polymer is a kind of simple preparation process, high efficiency, low toxicity gene carrier, is expected to in the future for clinical use gene therapy. DSP gene gun bullets prepared anti enzyme ability, is expected to improve the in vivo gene gun transfection efficiency.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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