乳酸菌胞外多糠的筛

发布时间：2018-06-06 09:59

本文选题：乳酸菌 + 胞外多糖　；参考：《内蒙古大学》2017年硕士论文

【摘要】：乳酸菌胞外多糖(Lactic acid bacteria exopolysaccharide,LAB EPS)具有多种生物活性,对其研究与开发具有重要的实际意义与良好的应用前景。本研究以实验室保存鉴定的乳酸菌为出发菌株,筛选能够增强细胞免疫活性的乳酸菌EPS,随后将其进行分离纯化及结构初步鉴定,在此基础上探讨其对免疫细胞活性及机体特异性免疫应答的影响。结果如下:1.对实验室保存鉴定的110株乳酸杆菌所产EPS进行测定,筛选10株高产EPS 乳酸杆菌,其中Lactobacillus harbinensis TCP086、Lactobaillus kefiri SXJ29和Lactobacillus casei SXJ30所产EPS可以增强巨噬细胞增殖、吞噬以及NO释放能力。2.将上述 Lactobacillus harbinensis TCP086、Lactobacillus kefiri SXJ29 和Lactobacillus casei SXJ30 所产 EPS 经过 DEAE-Sepharose Fast Flow 和 Sepharose CL-6B色谱柱进行分离纯化后获得主要均一组分:TCP086 EPS、SXJ29 EPS和SXJ30 EPS。紫外光谱和红外光谱扫描发现三种EPS均不含核酸和蛋白质,且均具有多糖的特征吸收峰,存在αα型吡喃糖环构型;多角度激光光散射仪检测其分子量分别为4.908 × 104 Da、3.423 × 104 Da和3.737 × 104 Da;离子色谱检测单糖组成发现,三种EPS均由氨基葡萄糖、阿拉伯糖、氨基半乳糖、半乳糖、葡萄糖、甘露糖以及葡萄糖醛酸等7种单糖组成,其中TCP086 EPS各主要组分摩尔比氨基葡萄糖:氨基半乳糖:甘露糖:葡萄糖醛酸约为2.6:1.6:1.2:1;SXJ29EPS各主要组分摩尔比葡萄糖:氨基半乳糖:氨基葡萄糖约为3.1:1:1;SXJ30 EPS各主要组分摩尔比葡萄糖:氨基葡萄糖:甘露糖约为1.4:1.1:1。3.进一步以纯化的TCP086 EPS、SXJ29 EPS和SXJ30 EPS刺激巨噬细胞,对其增殖能力、吞噬中性红能力、NO释放能力、细胞因子分泌以及表面分子CD40、CD80、CD86和MHC-Ⅱ表达进行检测后发现,巨噬细胞的免疫增强活性与EPS的浓度呈正相关,且SXJ30 EPS对巨噬细胞的免疫增强活性最强。4.在体外成功利用IL-4和GM-CSF诱导获得小鼠骨髓来源树突状细胞,显微镜下观察SXJ30EPS刺激小鼠BMDCs后,细胞突起增多,体积增大;流式细胞术和ELISA法检测后发现,SXJ30 EPS能显著增强小鼠BMDCs表面CD40、CD80、CD86和MHC-Ⅱ类分子的表达以及细胞因子(TNF-α、IL-6、IL-10和IL-12)的分泌。5.将SXJ30 EPS与OVA抗原免疫小鼠,发现SXJ30 EPS可以显著提高小鼠脾脏DCs表面CD40、CD80、CD86和MHC-Ⅱ类分子的表达量;与铝盐(Alum)佐剂相比,SXJ30 EPS可以显著提高小鼠血清中OVA特异性IgG抗体以及IgG抗体亚类IgG1、IgG2a和IgG2b的滴度;此外,SXJ30EPS还能显著促进脾脏T淋巴细胞增殖以及INF-γ和IL-4的分泌;显著提高脾脏IL-4+ CD4+ T细胞、IFN-γ+CD4+ T细胞以及IFN-γ+ CD8+ T细胞的比例,与Alum佐剂相比差异显著。上述结果提示:SXJ30 EPS在体外不仅可以增强巨噬细胞免疫活性,还能促进树突状细胞成熟和前炎性因子分泌。SXJ30 EPS可以显著提高小鼠对OVA抗原Th1型和Th2型免疫应答,其中主要以Th2型免疫应答为主。该研究为免疫增强剂的筛选和应用提供了可靠的理论指导和实验资料。
[Abstract]:Lactic acid bacteria exopolysaccharide (LAB EPS) has many biological activities. It has important practical significance and good application prospect for its research and development. In this study, lactic acid bacteria identified in the laboratory were used as the starting strain to screen the lactic acid bacteria EPS which could enhance the cellular immune activity, and then it was carried out. On the basis of the isolation and purification and preliminary identification of the structure, the effects on the immune cell activity and the specific immune response of the body were investigated. The results were as follows: 1. the EPS produced by 110 Lactobacillus strains preserved in the laboratory was determined, and 10 strains of high yield EPS lactobacillus were screened, including Lactobacillus harbinensis TCP086, Lactobaillus kefiri SXJ29. And EPS produced by Lactobacillus and casei SXJ30 can enhance macrophage proliferation, phagocytosis, and NO release ability.2. to isolate and purify the above Lactobacillus harbinensis TCP086, Lactobacillus kefiri SXJ29 and purified chromatographic column. A homogeneous component: TCP086 EPS, SXJ29 EPS and SXJ30 EPS. UV and IR spectrum scanning found that all three kinds of EPS have no nucleic acid and protein, and all have the characteristic absorption peaks of polysaccharide, and there is alpha alpha type Piran ring configuration, and the molecular weight of the multi angle laser light scattering instrument is 4.908 x 104 Da, 3.423 x 104 Da and 3.737 x 104 Da respectively. The determination of monosaccharide composition by ion chromatography shows that three kinds of EPS are composed of 7 kinds of monosaccharides, such as glucosamine, Arabia sugar, amino galactose, galactose, glucose, mannose, and glucuronic acid. The main components of TCP086 EPS are amino glucose: mannose: glucuronic acid is about 2.6:1.6:1.2:1; SXJ29EPS Mole ratio glucose: amino galactose: amino galactose: Glucosamine is about 3.1:1:1; SXJ30 EPS is the main component of glucose: Glucosamine: Glucosamine: mannose is about 1.4:1.1:1.3. to further stimulate macrophage by purified TCP086 EPS, SXJ29 EPS and SXJ30 EPS, to its proliferating ability, phagocytosis of neutral red, NO release capacity, cytokine The secretion and the expression of surface molecules CD40, CD80, CD86 and MHC- II showed that the immune enhancement activity of macrophages was positively related to the concentration of EPS, and the strongest immune activity of SXJ30 EPS on macrophage was the strongest.4. in vitro, which was successfully used to induce mouse bone marrow derived dendritic cells by IL-4 and GM-CSF in vitro. Under microscope, the SXJ30EP was observed. After S stimulated BMDCs, the cell protruding increased and the volume increased. After flow cytometry and ELISA assay, it was found that SXJ30 EPS could significantly enhance the expression of CD40, CD80, CD86 and MHC- II molecules on the BMDCs surface of mice and the secretion of cytokines (TNF- alpha, IL-6, etc.). The expression of CD40, CD80, CD86 and MHC- II molecules on the DCs surface of the spleen was improved. Compared with the aluminum salt (Alum) adjuvant, SXJ30 EPS could significantly increase the OVA specific IgG antibody and IgG antibody subclass IgG1, IgG antibody and the titer. The proportion of spleen IL-4+ CD4+ T cells, IFN- gamma +CD4+ T cells and IFN- gamma + CD8+ T cells was significantly different from Alum adjuvant. These results suggest that SXJ30 EPS in vitro can not only enhance macrophage immunological activity, but also promote dendritic cell maturation and proinflammatory cytokines secretion. The immune response of OVA antigen Th1 and Th2 type is mainly based on Th2 type immune response. This study provides reliable theoretical guidance and experimental data for screening and application of immune enhancers.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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