本文選題：核酸疫苗 + 殼聚糖�。� 參考：《東北師范大學(xué)》2011年碩士論文

【摘要】：殼聚糖(CS)是多糖中唯一天然堿性陽離子多聚糖。近年來,因其生物可降解性、生物粘附性、良好的生物相容性,以及較低的細(xì)胞毒性而得到廣泛應(yīng)用;殼聚糖在弱酸性溶液中氨基發(fā)生質(zhì)子化,可以與表面帶有負(fù)電荷的核酸通過靜電作用形成納米級(jí)粒子。提高核酸利用度,保護(hù)核酸不被酶降解等眾多特性,使其作為優(yōu)良的核酸載體受到廣泛關(guān)注。 本文使用復(fù)凝法將殼聚糖與白色念珠菌熱休克蛋白90重組質(zhì)粒(DNA_(ch90))形成納米粒子,并對(duì)其在動(dòng)物實(shí)驗(yàn)中的緩釋作用進(jìn)行初步探討。 1、多種因素對(duì)納米粒子大小及電位產(chǎn)生影響實(shí)驗(yàn)結(jié)果表明N/P比值、DNA_(ch90)濃度、pH值、Na_2SO_4濃度、水浴溫度與納米粒子粒徑大小呈正相關(guān);N/P比值和DNA_(ch90)濃度與納米粒子表面電位呈正相關(guān);pH值、Na_2SO_4濃度和水浴溫度與顆粒表面電位呈負(fù)相關(guān)。以此優(yōu)化條件制備CS-DNA_(ch90)納米粒子。 2、初步探究CS-DNA_(ch90)納米粒子的理化性質(zhì)和相關(guān)特性 測(cè)定納米粒子的形態(tài)、粒徑大小和分布、表面電位。結(jié)果顯示制備的納米粒子大小均一,直徑在166nm左右;分散度良好;表面電位也為正值。 測(cè)量納米粒子的包封率和載藥量。結(jié)果顯示其包封率高于90%,載藥量大于30%;考量了CS濃度對(duì)包封率的影響。CS濃度與包封率正相關(guān)。凝膠阻滯分析實(shí)驗(yàn)說明粒子表面帶有正電荷,殼聚糖將DNA_(ch90)包裹在顆粒中心;酶保護(hù)實(shí)驗(yàn)進(jìn)一步證明,CS-DNA_(ch90)納米粒抵御DNA_(ch90)被酶解。 3、CS-DNA ch90納米粒子在動(dòng)物體內(nèi)對(duì)核酸疫苗的緩釋作用 以CS-DNA_(ch90)納米粒子為實(shí)驗(yàn)材料,采用皮下兩點(diǎn)注射的方式免疫ICR小鼠,免疫兩次,間隔時(shí)間為兩周。免疫小鼠抗血清與R-Hsp90蛋白Western Blot結(jié)果表明,CS-DNA_(ch90)納米粒子能夠使免疫小鼠產(chǎn)生抗R-Hsp90特異性抗體。ELISA檢測(cè)免疫小鼠抗血清抗R-Hsp90特異性抗體IgG、IgG1及IgG2b含量,CS-DNA_(ch90)納米粒子組的抗體含量遠(yuǎn)遠(yuǎn)低于DNA ch90組小鼠抗體含量(P0.01),該結(jié)果表明CS-DNA_(ch90)納米粒子在動(dòng)物體內(nèi)對(duì)核酸疫苗起到了明顯的緩釋作用。
[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.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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本文編號(hào)：1913088