【摘要】：肺炎克雷伯菌（Klebsiella Pneumoniae）是人和動(dòng)物呼吸道、消化道的條件致病菌。菌毛是肺炎克雷伯菌的重要致病因子，其通過(guò)菌毛尖端粘附素（adhesin）的粘附作用定植在宿主黏膜表面繼而進(jìn)行感染。 黏膜是機(jī)體抵抗病原體入侵的第一道屏障，由黏膜進(jìn)行免疫接種可有效誘導(dǎo)局部和全身免疫應(yīng)答。而絕大多數(shù)抗原經(jīng)黏膜供給時(shí)免疫原性弱且應(yīng)答持續(xù)時(shí)間短，因此，一種高效、安全、無(wú)副作用的黏膜佐劑對(duì)于黏膜免疫十分重要。 多糖是生命有機(jī)體的重要組成成分，具有多種生物學(xué)功能，其中最主要的是免疫調(diào)節(jié)作用。腸道黏膜是口服多糖發(fā)揮局部免疫調(diào)節(jié)作用從而影響整體免疫的主要場(chǎng)所。多糖的副作用小，不易產(chǎn)生耐受，將其開(kāi)發(fā)為新型佐劑，，具有十分廣闊的應(yīng)用前景。 首先，本研究采用分子生物學(xué)技術(shù)在大腸桿菌BL21Codon Plus（DE3）中成功誘導(dǎo)表達(dá)肺炎克雷伯氏菌Ⅰ型菌毛粘附蛋白K（fimK），并以fimK作為抗原制備多克隆抗體。 然后對(duì)茯苓多糖（Pachymaran）和枸杞多糖(lyciumbarbarum polysaccharides）對(duì)免疫抑制小鼠的免疫增強(qiáng)和腸道黏膜免疫調(diào)節(jié)作用進(jìn)行研究，結(jié)果顯示，兩種多糖均可提高免疫抑制小鼠脾臟指數(shù)，增強(qiáng)腹腔巨噬細(xì)胞吞噬功能，調(diào)節(jié)免疫抑制小鼠脾臟CD3+CD4+/CD3+CD8+細(xì)胞亞群比例及腸派氏結(jié)T、B淋巴細(xì)胞比例和血清及小腸組織中IL-12、IL-4、IFN-γ細(xì)胞因子水平。證明枸杞多糖和茯苓多糖均對(duì)免疫抑制小鼠具有免疫增強(qiáng)作用，并對(duì)腸道黏膜系統(tǒng)具有免疫調(diào)節(jié)作用，且茯苓多糖的免疫調(diào)節(jié)效應(yīng)顯著優(yōu)于枸杞多糖。 進(jìn)一步探討多糖在黏膜免疫中的佐劑效果，本研究制備了PLGA包裹fimK微球作為口服免疫的抗原并以茯苓多糖作為黏膜佐劑進(jìn)行免疫，并通過(guò)肺炎克雷伯菌攻毒保護(hù)性實(shí)驗(yàn)，評(píng)價(jià)疫苗的保護(hù)效力。結(jié)果顯示，多糖佐劑組血清特異性IgG、腸黏膜特異性IgA水平均顯著高于其他組，能夠顯著增強(qiáng)體液免疫應(yīng)答。通過(guò)檢測(cè)脾臟和腸派氏結(jié)CD3+CD4+/CD3+CD8+淋巴細(xì)胞比例和血清及腸組織中IFN-γ和IL-4細(xì)胞因子水平，顯示CD3+CD4+/CD3+CD8+細(xì)胞比值顯著增大，提示機(jī)體免疫應(yīng)答主要是CD4+表型T細(xì)胞輔助參與調(diào)節(jié)，同時(shí)IL-4細(xì)胞因子水平顯著升高，提示茯苓多糖為佐劑誘導(dǎo)的免疫應(yīng)答偏向Th2型，并能夠?qū)π∈笤谥滤佬苑窝卓死撞旯ザ局衅鸬奖Ｗo(hù)作用。 本研究探討了茯苓多糖在貂源肺炎克雷伯菌菌毛粘附蛋白黏膜免疫中的佐劑效應(yīng)，結(jié)果顯示茯苓多糖能夠增強(qiáng)小鼠局部黏膜免疫應(yīng)答和系統(tǒng)免疫反應(yīng)，并對(duì)致死性肺炎克雷伯菌株的攻毒具有保護(hù)作用。表明了茯苓多糖作為免疫增強(qiáng)劑具有開(kāi)發(fā)為黏膜佐劑的潛力。
[Abstract]:Klebsiella pneumoniae (Klebsiella Pneumoniae) is a conditioned pathogen of respiratory tract and digestive tract in humans and animals. Pili is an important pathogenic factor of Klebsiella pneumoniae. It is infected on the surface of the host mucosa by the adhesion of pili tip adhesion hormone (adhesin). Mucous membrane is the first barrier against pathogen invasion. Local and systemic immune responses can be effectively induced by mucosal immunization. However, most antigens are weak in immunogenicity and short in response duration. Therefore, a highly efficient, safe and non-side-effect mucosal adjuvant is very important for mucosal immunity. Polysaccharide is an important component of living organisms and has a variety of biological functions, the most important of which is immune regulation. Intestinal mucosa is the main site in which oral polysaccharides play a role in local immune regulation and thus affect the overall immunity. Polysaccharide has a wide application prospect because of its small side effect and difficult to be tolerated as a new adjuvant. Firstly, molecular biology technique was used to induce the expression of Klebsiella pneumoniae type I pili adhesion protein (K (fimK),) in Escherichia coli BL21Codon Plus (DE3) and the polyclonal antibody was prepared by using fimK as antigen. Then the immune enhancement and intestinal mucosal immunomodulation of Poria cocos polysaccharide (Pachymaran) and Lycium barbarum polysaccharide (lyciumbarbarum polysaccharides) in immunosuppressive mice were studied. The results showed that the two polysaccharides could increase the spleen index of immunosuppressive mice. The phagocytic function of peritoneal macrophages was enhanced, and the ratio of CD3 CD4 / CD3 CD8 cell subsets in spleen, the ratio of B lymphocytes in intestinal Pak's knot and the level of IL-12,IL-4,IFN- 緯 cytokines in serum and small intestine tissue were regulated in immunosuppressive mice. The results showed that both Lycium barbarum polysaccharides and Poria cocos polysaccharides had immune enhancement effect on immunosuppressive mice and immune regulation on intestinal mucosal system, and the immune regulation effect of Poria cocos polysaccharide was significantly better than that of Lycium barbarum polysaccharide. To further study the adjuvant effect of polysaccharides in mucosal immunity, PLGA coated fimK microspheres were prepared as oral immunity antigen and Poria cocos polysaccharide as mucosal adjuvant. To evaluate the protective efficacy of vaccines. The results showed that the levels of serum specific IgG, mucosal specific IgA in polysaccharide adjuvant group were significantly higher than those in other groups, and could significantly enhance humoral immune response. The ratio of IFN- 緯 and IL-4 cytokines in serum and intestinal tissue was significantly increased by detecting the ratio of CD3 CD4 / CD3 CD8 lymphocytes in spleen and intestinal node and the levels of IFN- 緯 and IL-4 cytokines in serum and intestinal tissue. It suggested that the immune response was mainly mediated by CD4 phenotypic T cells, while the level of IL-4 cytokines was significantly increased, suggesting that the immune response induced by Poria cocos polysaccharide as adjuvant tended to Th2 type. It can protect mice against Klebsiella pneumoniae. In this study, we studied the adjuvant effect of Poria cocos polysaccharide in mucosal immunity of Klebsiella mink pneumoniae pili adhesion protein. The results showed that Poria cocos polysaccharide could enhance local mucosal immune response and systemic immune response in mice. It also has protective effect on the attack of Klebsiella pneumoniae. It is suggested that Poria cocos polysaccharide as an immunoenhancer has the potential to be developed as a mucosal adjuvant.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S858.92

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