壳聚糖—白念菌热休克蛋白90核酸疫苗纳米粒子的制备及其缓释作用初探

发布时间：2018-05-20 04:01

本文选题：核酸疫苗 + 壳聚糖　；参考：《东北师范大学》2011年硕士论文

【摘要】：壳聚糖(CS)是多糖中唯一天然碱性阳离子多聚糖。近年来,因其生物可降解性、生物粘附性、良好的生物相容性,以及较低的细胞毒性而得到广泛应用;壳聚糖在弱酸性溶液中氨基发生质子化,可以与表面带有负电荷的核酸通过静电作用形成纳米级粒子。提高核酸利用度,保护核酸不被酶降解等众多特性,使其作为优良的核酸载体受到广泛关注。本文使用复凝法将壳聚糖与白色念珠菌热休克蛋白90重组质粒(DNA_(ch90))形成纳米粒子,并对其在动物实验中的缓释作用进行初步探讨。 1、多种因素对纳米粒子大小及电位产生影响实验结果表明N/P比值、DNA_(ch90)浓度、pH值、Na_2SO_4浓度、水浴温度与纳米粒子粒径大小呈正相关;N/P比值和DNA_(ch90)浓度与纳米粒子表面电位呈正相关;pH值、Na_2SO_4浓度和水浴温度与颗粒表面电位呈负相关。以此优化条件制备CS-DNA_(ch90)纳米粒子。 2、初步探究CS-DNA_(ch90)纳米粒子的理化性质和相关特性测定纳米粒子的形态、粒径大小和分布、表面电位。结果显示制备的纳米粒子大小均一,直径在166nm左右;分散度良好;表面电位也为正值。测量纳米粒子的包封率和载药量。结果显示其包封率高于90%,载药量大于30%;考量了CS浓度对包封率的影响。CS浓度与包封率正相关。凝胶阻滞分析实验说明粒子表面带有正电荷,壳聚糖将DNA_(ch90)包裹在颗粒中心;酶保护实验进一步证明,CS-DNA_(ch90)纳米粒抵御DNA_(ch90)被酶解。 3、CS-DNA ch90纳米粒子在动物体内对核酸疫苗的缓释作用以CS-DNA_(ch90)纳米粒子为实验材料,采用皮下两点注射的方式免疫ICR小鼠,免疫两次,间隔时间为两周。免疫小鼠抗血清与R-Hsp90蛋白Western Blot结果表明,CS-DNA_(ch90)纳米粒子能够使免疫小鼠产生抗R-Hsp90特异性抗体。ELISA检测免疫小鼠抗血清抗R-Hsp90特异性抗体IgG、IgG1及IgG2b含量,CS-DNA_(ch90)纳米粒子组的抗体含量远远低于DNA ch90组小鼠抗体含量(P0.01),该结果表明CS-DNA_(ch90)纳米粒子在动物体内对核酸疫苗起到了明显的缓释作用。
[Abstract]:Chitosan (CS) is the only natural alkaline cationic polysaccharide in polysaccharides. In recent years, it has been widely used because of its biodegradability, bioadhesion, good biocompatibility and low cytotoxicity. Nanocrystalline particles can be formed by electrostatic interaction with negatively charged nucleic acids on the surface. Increasing the utilization of nucleic acid and protecting nucleic acid from degradation by enzyme makes it a good carrier of nucleic acid. In this paper, chitosan and Candida albicans heat shock protein 90 recombinant plasmid DNASch 90) were used to form nanoparticles, and their slow release effects in animal experiments were preliminarily discussed. 1. The experimental results of the effects of various factors on the size and potential of the nanoparticles showed that the concentration of N- / P ratio and the concentration of DNA tippon _ 90 (pH value) were higher than the concentration of Na _ 2SO _ 4. There was a positive correlation between the water bath temperature and the particle size, the ratio of N / P and DNA tip-90) and the surface potential of the nanoparticles. There was a negative correlation between the concentration of Na _ 2SO _ 4 and the water bath temperature and the surface potential of the nanoparticles. CS-DNA tip-90) nanoparticles were prepared under the optimized conditions. 2. Preliminary study on the physicochemical properties and related properties of CS-DNA aphach90) nanoparticles The morphology, particle size and distribution, surface potential of nanoparticles were measured. The results show that the size of the prepared nanoparticles is uniform, the diameter is about 166nm, the dispersion is good, and the surface potential is positive. The encapsulation efficiency and drug loading of nanoparticles were measured. The results showed that the entrapment efficiency was higher than 90 and the drug load was more than 30. The effect of CS concentration on the encapsulation efficiency was considered. The concentration of CS was positively correlated with the encapsulation efficiency. Gel block analysis showed that the surface of the particles was positively charged, and chitosan encapsulated the DNA tipped ch90 in the center of the particles, while the enzyme protection experiments further proved that the CS-DNA tich90 nanoparticles were hydrolyzed against DNASch _ (90). Sustained release of CS-DNA ch90 nanoparticles on Nucleic Acid Vaccine in Animals ICR mice were immunized by subcutaneous two-point injection with CS-DNA tippon 90 nanoparticles as the experimental material. The time interval was two weeks. The results of anti-serum and Western Blot of R-Hsp90 protein in immunized mice showed that CS-DNA tich90) nanoparticles could make immunized mice produce specific antibody against R-Hsp90. Elisa was used to detect the content of anti-R-Hsp90 specific antibody IgG1 and IgG2b in anti-serum of immunized mice and the antibody content of CS-DNA tich90) nanoparticles in immunized mice was determined by enzyme-linked immunosorbent assay (Elisa). The amount of antibody in DNA ch90 group was much lower than that in DNA ch90 group. The results showed that CS-DNA tich90) nanoparticles had an obvious slow-release effect on nucleic acid vaccine in animals.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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