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剂效应
发布时间：2019-02-14 09:31
【摘要】：肺炎克雷伯菌（Klebsiella Pneumoniae）是人和动物呼吸道、消化道的条件致病菌。菌毛是肺炎克雷伯菌的重要致病因子，其通过菌毛尖端粘附素（adhesin）的粘附作用定植在宿主黏膜表面继而进行感染。 黏膜是机体抵抗病原体入侵的第一道屏障，由黏膜进行免疫接种可有效诱导局部和全身免疫应答。而绝大多数抗原经黏膜供给时免疫原性弱且应答持续时间短，因此，一种高效、安全、无副作用的黏膜佐剂对于黏膜免疫十分重要。 多糖是生命有机体的重要组成成分，具有多种生物学功能，其中最主要的是免疫调节作用。肠道黏膜是口服多糖发挥局部免疫调节作用从而影响整体免疫的主要场所。多糖的副作用小，不易产生耐受，将其开发为新型佐剂，，具有十分广阔的应用前景。 首先，本研究采用分子生物学技术在大肠杆菌BL21Codon Plus（DE3）中成功诱导表达肺炎克雷伯氏菌Ⅰ型菌毛粘附蛋白K（fimK），并以fimK作为抗原制备多克隆抗体。 然后对茯苓多糖（Pachymaran）和枸杞多糖(lyciumbarbarum polysaccharides）对免疫抑制小鼠的免疫增强和肠道黏膜免疫调节作用进行研究，结果显示，两种多糖均可提高免疫抑制小鼠脾脏指数，增强腹腔巨噬细胞吞噬功能，调节免疫抑制小鼠脾脏CD3+CD4+/CD3+CD8+细胞亚群比例及肠派氏结T、B淋巴细胞比例和血清及小肠组织中IL-12、IL-4、IFN-γ细胞因子水平。证明枸杞多糖和茯苓多糖均对免疫抑制小鼠具有免疫增强作用，并对肠道黏膜系统具有免疫调节作用，且茯苓多糖的免疫调节效应显著优于枸杞多糖。 进一步探讨多糖在黏膜免疫中的佐剂效果，本研究制备了PLGA包裹fimK微球作为口服免疫的抗原并以茯苓多糖作为黏膜佐剂进行免疫，并通过肺炎克雷伯菌攻毒保护性实验，评价疫苗的保护效力。结果显示，多糖佐剂组血清特异性IgG、肠黏膜特异性IgA水平均显著高于其他组，能够显著增强体液免疫应答。通过检测脾脏和肠派氏结CD3+CD4+/CD3+CD8+淋巴细胞比例和血清及肠组织中IFN-γ和IL-4细胞因子水平，显示CD3+CD4+/CD3+CD8+细胞比值显著增大，提示机体免疫应答主要是CD4+表型T细胞辅助参与调节，同时IL-4细胞因子水平显著升高，提示茯苓多糖为佐剂诱导的免疫应答偏向Th2型，并能够对小鼠在致死性肺炎克雷伯菌株攻毒中起到保护作用。 本研究探讨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剂效应，结果显示茯苓多糖能够增强小鼠局部黏膜免疫应答和系统免疫反应，并对致死性肺炎克雷伯菌株的攻毒具有保护作用。表明了茯苓多糖作为免疫增强剂具有开发为黏膜佐剂的潜力。
[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- 纬 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- 纬 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- 纬 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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.92
本文编号：2422081
[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- 纬 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- 纬 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- 纬 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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.92
【参考文献】
相关期刊论文 前10条
1 管远红;傅宏庆;邹志恒;韦启鹏;;3种植物多糖对鸡外周血和脾脏淋巴细胞体外增殖作用的影响[J];中国畜牧兽医;2014年12期
2 杨姗姗;张秀娟;;半枝莲多糖对荷瘤小鼠T细胞免疫功能的影响[J];河南农业科学;2014年06期
3 韩洁;岳文鹏;何光志;田维毅;;八种中药多糖对小鼠巨噬细胞释放细胞因子的影响[J];现代免疫学;2011年06期
4 贾薇;樊华;;姬松茸多糖组分对小鼠巨噬细胞作用研究[J];食品科学;2011年11期
5 郝冉;王正明;查学强;潘利华;罗建平;;霍山石斛多糖的肠黏膜免疫调节活性及在小肠中的吸收分布[J];食品科学;2014年09期
6 刘梁;孙维矿;赵玲;李赫宇;陈新;;米糠多糖的免疫增强作用[J];食品研究与开发;2014年22期
7 胡庭俊,陈炅然,程富胜,孟聚诚,高芳,张霞,高艳艳;蕨麻多糖对小鼠血清中三种细胞因子水平的影响[J];中国兽医科技;2005年08期
8 胡新俊;李春香;王广;马淑霞;李丽秋;杨景云;;马齿苋多糖对肠道微生态失调小鼠的调整作用研究[J];中国微生态学杂志;2010年09期
9 王青;胡明华;董燕;潘华新;周联;王培训;;茯苓多糖对免疫抑制小鼠粘膜淋巴组织及脾脏中CD3~+和CD19~+细胞变化的影响[J];中国免疫学杂志;2011年03期
10 张训海;王德云;胡元亮;孙峻岭;;黄芪多糖对鸡体液免疫增强作用[J];中国兽医学报;2009年03期
相关博士学位论文 前1条
1 李扬;肺炎克雷伯菌免疫相关表位筛选及其实验免疫研究[D];吉林大学;2010年
本文编号：2422081
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2422081.html
