本文選題：中性糖　切入點(diǎn)：兩性葡聚糖　出處：《福州大學(xué)》2006年碩士論文

【摘要】： 多糖屬于作為T細(xì)胞非依賴性抗原,主要通過(guò)增強(qiáng)機(jī)體的非特異性免疫功能以抵御細(xì)菌、病毒和寄生蟲等病原微生物的侵襲,其沒有免疫記憶效應(yīng),無(wú)法直接誘導(dǎo)機(jī)體產(chǎn)生高效價(jià)特異性IgG抗體,因而在疫苗的制備上通常需將多糖連接于一定得載體上形成偶聯(lián)物,刺激機(jī)體產(chǎn)生高親和力IgG抗體。而兩性葡聚糖與一般多糖不同,它能與抗原遞呈細(xì)胞進(jìn)而激活T細(xì)胞的免疫調(diào)節(jié)機(jī)制,之后誘導(dǎo)機(jī)體產(chǎn)生IgG亞類抗體。本課題著重討論了兩性葡聚糖體液免疫應(yīng)答和調(diào)節(jié)效應(yīng)的特異性及其產(chǎn)生機(jī)制。分別以中性糖、兩性葡聚糖、糖蛋白為抗原,比較它們?cè)诳贵w水平和細(xì)胞水平上的體液免疫應(yīng)答和免疫調(diào)節(jié)效應(yīng),從而探尋兩性葡聚糖在免疫行為上的特性。 本課題采用硫酸酯化法修飾葡糖胺制備帶有陰性基團(tuán)和陽(yáng)性基團(tuán)的兩性葡聚糖,采用雙環(huán)氧試劑(1,4-二丁醇二縮水甘油醚)交聯(lián)蛋白載體制備糖蛋白。相關(guān)檢測(cè)結(jié)果顯示,兩性葡聚糖硫酸根濃度為36. 14 ug/ml,取代度為0.90,而氨基氮含量為35.04ug/ml,取代度為0.91,制得帶有正、負(fù)電荷的基團(tuán)的兩性葡聚糖。糖蛋白交聯(lián)產(chǎn)物電泳及HPLC檢測(cè)結(jié)果表明,經(jīng)雙環(huán)氧試劑交聯(lián)以及分離純化等步驟獲得了純度較高的糖蛋白分子。 采用中性葡聚糖(分子量分別為2萬(wàn),27萬(wàn))、兩性葡聚糖、糖蛋白三種抗原體外刺激的細(xì)胞增殖以及細(xì)胞因子表達(dá)的結(jié)果表明,兩性葡聚糖能顯著促進(jìn)不同糖免疫后的細(xì)胞增殖,并刺激細(xì)胞表達(dá)TLR4、TLR9、TNFα等細(xì)胞因子。中性糖、糖蛋白、兩性葡聚糖刺激機(jī)體形成抗體生成細(xì)胞的實(shí)驗(yàn)結(jié)果顯示,兩性葡聚糖除了能刺激細(xì)胞表達(dá)IgM亞類抗體外還能刺激其表達(dá)IgG亞類抗體,而中性多糖則只能表達(dá)IgM亞類抗體。 在抗體水平上通過(guò)ELISA檢測(cè)中性糖、兩性葡聚糖、糖蛋白抗體效價(jià)以及相對(duì)親和常數(shù),結(jié)果顯示兩性葡聚糖與糖蛋白有相似的免疫應(yīng)答效應(yīng),都能刺激機(jī)體表達(dá)糖特異性的IgG類抗體,且兩性葡聚糖抗原與其他糖刺激的抗體表現(xiàn)出較強(qiáng)的交叉反應(yīng)。 實(shí)驗(yàn)結(jié)果說(shuō)明兩性葡聚糖具有特異的免疫應(yīng)答和調(diào)節(jié)效應(yīng),為多糖疫苗的設(shè)計(jì)與開發(fā)提供一個(gè)新的方向。
[Abstract]:Polysaccharides, as T-cell independent antigens, can resist the invasion of bacteria, viruses, parasites and other pathogenic microorganisms by enhancing the body's non-specific immune function, and it has no immune memory effect.It is impossible to directly induce the production of high titer specific IgG antibodies, so it is usually necessary to attach polysaccharides to a certain carrier to form conjugates in order to stimulate the production of high affinity IgG antibodies.The amphoteric dextran is different from other polysaccharides in that it can activate the immune regulation mechanism of T cells with antigen-presenting cells and then induce the production of IgG subclass antibodies.The purpose of this paper is to discuss the specificity and mechanism of humoral immune response and regulation of amphoteric dextran.Neutral sugar, amphoteric dextran and glycoprotein were used as antigens to compare their humoral immune responses and immunomodulatory effects at the antibody level and cell level, respectively, in order to explore the immunological behavior of amphoteric dextran.In this study, glucosamine was modified by sulfate to prepare amphoteric dextran with negative and positive groups.Correlation analysis showed that the concentration of amphoteric dextran sulfate was 36. 5%.The degree of substitution is 0.90, while the content of amino nitrogen is 35.04 渭 / ml, and the degree of substitution is 0.91.Amphoteric dextran with positive and negative charge groups is prepared.The results of electrophoretic analysis and HPLC analysis showed that high purity glycoprotein molecules were obtained by double epoxide crosslinking and separation and purification.The results of cell proliferation and cytokine expression stimulated by neutral dextran (molecular weight 20, 270000), amphoteric dextran and glycoprotein in vitro showed that amphoteric dextran could significantly promote cell proliferation after different glucose immunization.TLR4, TLR9, TNF 偽 and other cytokines were also stimulated.Neutral sugar, glycoprotein and amphoteric dextran stimulated the formation of antibody producing cells. The results showed that amphoteric dextran not only stimulated the expression of IgM subclass antibody, but also stimulated the expression of IgG subclass antibody.Neutral polysaccharides can only express IgM subclass antibodies.The antibody titers of neutral sugar, amphoteric dextran, glycoprotein antibody and relative affinity constant were detected by ELISA at the antibody level. The results showed that amphoteric dextran and glycoprotein had similar immune response effects.Amphoteric dextran antigen showed strong cross reaction with other sugar stimulated antibodies.The results show that amphoteric dextran has a specific immune response and regulatory effect, which provides a new direction for the design and development of polysaccharide vaccine.
【學(xué)位授予單位】：福州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392.1

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