乳酸杆菌DM9811肽聚糖的分离提取及免疫活性的研究

发布时间：2018-02-07 10:05

本文关键词： 乳酸杆菌肽聚糖提取免疫活性　出处：《大连医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景乳酸杆菌是人体中一种非常重要的益生菌,其生物学作用包括生物拮抗,营养作用,免疫调节,抗肿瘤,抗衰老等作用。乳酸杆菌通过其特异的细胞组分与宿主的免疫细胞相互作用而发挥免疫调节功能。肽聚糖(peptidoglycan)也称黏肽(mucopeptide),是乳酸杆菌细胞壁结构和理化性质的主要功能性物质。肽聚糖的基本结构是由N-乙酰葡萄糖(GlcNAc)和N-乙酰胞壁酸(MurNAc)通过β-1,4糖苷键连接,每个N-乙酰胞壁酸(MurNAc)又连接一个四肽侧链,每个四肽侧链又通过一个五聚甘氨酸组成的肽交联桥(peptide across bridge)连接,肽聚糖是一类由共价键连接,包围整个细菌细胞壁的囊状大分子(sac-liked macromolecule)。近年来,肽聚糖的生物活性一直受到关注。 Toll样受体(Toll like receptor,TLR)属于膜识别受体,是近年发现的一种膜表面受体。TLR样受体通过上调抗原递呈细胞表面的共刺激分子及抗原递呈细胞分泌的细胞因子,诱导T和B淋巴细胞向效应T和B淋巴细胞分化,进而调节获得性免疫。TLRs可以识别微生物所特有的保守结构,这类保守结构称为微生物相关保守分子(pathogen associated molecular pattern,PAMP)。肽聚糖是乳酸杆菌含量最丰富的MAMP之一,可能通过TLRs的识别而发挥免疫作用。目的本文以大连医科大学微生物教研室分离的一株乳酸杆菌DM9811为研究对象,通过提取其细胞壁的主要成分肽聚糖,观察肽聚糖对小鼠巨噬细胞Ana-1的免疫调节作用,为进而探索肽聚糖发挥免疫调节作用的分子机制提供实验基础。方法 1、肽聚糖的分离纯化收集培养48小时的乳酸杆菌DM9811菌体,经10%三氯乙酸煮沸20分钟和含有4%SDS的PBS煮沸30分钟后,再用胰酶消化48小时,离心收集的沉淀即为肽聚糖粗提取物。肽聚糖粗提取物进一步溶菌酶酶解后,经葡聚糖凝胶SephacrylS-300HR进行分离和部分纯化。 2、肽聚糖鉴定应用苯酚-硫酸法测定总糖含量,对二甲氨基苯甲醛显色联合溶菌酶水解实验测定N-乙酰葡萄糖,测定酶解过程中溶液吸光度的变化情况来鉴定肽聚糖。 3、肽聚糖的免疫活性肽聚糖处理小鼠巨噬细胞Ana-1前后,分别1)应用酶联免疫吸附剂测定法(enzyme linked immunosorbent assay, ELISA)检测培养上清中的IL-10,IL-12和TNF-α的蛋白水平;2)在活细胞染料羧基荧光素乙酰乙酸琥珀酸亚胺酯(CFSE)标记小鼠巨噬细胞Ana-1后,流式细胞术检测小鼠巨噬细胞Ana-1的增殖分裂;3)流式细胞术检测肽小鼠巨噬细胞Ana-1细胞表面TLR2和TLR4的表达。结果 1、应用三氯乙酸和SDS煮沸以及胰酶酶解得到肽聚糖,经苯酚-硫酸法,对二甲氨基苯甲醛显色,溶菌酶水解实验鉴定,凝胶过滤层析显示从乳酸杆菌DM9811获得了分子量相对均一的肽聚糖。 2、ELISA结果显示所提取的肽聚糖可以促进小鼠巨噬细胞Ana-1分泌IL-10,IL-12,而低浓度的肽聚糖可以促进TNF-α分泌;流式细胞技术检测结果显示,与对照组相比,肽聚糖处理的巨噬细胞Ana-1分裂代数明显增加,同时细胞表面TLR2和TLR4(主要是TLR4)表达均显著提高。结论乳酸杆菌DM9811肽聚糖提取物表现在体外激活小鼠巨噬细胞Ana-1的免疫活性。
[Abstract]:background
Lactobacillus is one of the most important probiotics in human body, its biological effects including biological antagonism, nutrition, immune regulation, anti-tumor, anti-aging effect. Lactobacillus by its specific cellular components and the interaction of host immune cells and immune regulating function.
Peptidoglycan (peptidoglycan) also called peptidoglycan (mucopeptide), is the main function of lactic acid bacteria cell wall structure and physicochemical properties of the material. The basic structure of peptidoglycan by N- acetyl glucose (GlcNAc) and N- n-acetylmuramicacid (MurNAc) connected by -1,4 beta glycosidic bond, each N- n-acetylmuramicacid (MurNAc) with a four peptide side chain, each of the four peptide side chains and by a peptide consisting of five poly glycine called (peptide across bridge) is a kind of connection, peptidoglycan connected by covalent bond, cystic molecules surrounding the whole bacterial cell wall (sac-liked macromolecule). In recent years, peptide the biological activity of chitosan has been of great concern.
Toll like receptors (Toll like receptor, TLR) belongs to membrane recognition receptor, is a recently identified membrane receptor.TLR like receptor through upregulation of antigen-presenting cell surface costimulatory molecules and antigen-presenting cells secrete cytokines that induce T and B lymphocytes to the effects of T and B lymphocyte differentiation, and then adjust the gain immunity.TLRs can keep a special structure of microbial recognition, this kind of conservative structure called the microbe (pathogen associated molecular pattern conservative, PAMP).
Peptidoglycan is one of the most abundant MAMP lactobacilli, which may play an immune role through the identification of TLRs.
objective
A strain of Lactobacillus DM9811 in the Department of Microbiology Dalian Medical University separation as the research object, the main component of peptidoglycan extracted from the cell wall peptidoglycan, observe the immune regulating effect of mouse macrophage Ana-1, provide experimental basis for the molecular mechanism play a role in immune regulation and then explore the peptidoglycan.
Method
1, separation and purification of peptidoglycan
Collect the culture of Lactobacillus DM9811 was 48 hours, 30 minutes after the 10% three chloroacetic acid boiling for 20 minutes and 4%SDS containing PBS after boiling, and then trypsin for 48 hours, the precipitate is collected by centrifugation. The crude extract peptidoglycan peptidoglycan crude extract further lysozyme after enzymatic hydrolysis by Sephadex SephacrylS-300HR were separated and part.
2, peptidoglycan identification
The total sugar content was determined by phenol sulfuric acid method. The N- acetyl glucose was determined by the hydrolysis of two methylamino benzaldehyde combined with lysozyme, and the change of solution absorbency in the process of enzymatic hydrolysis was determined to identify peptidoglycan.
3, the immunological activity of peptidoglycan
Before and after the treatment of peptidoglycan of mouse macrophage Ana-1 1, respectively) by enzyme linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) in culture supernatants was measured by IL-10, IL-12 and protein levels of TNF- alpha; 2) in live cell dye carboxyfluorescein diacetate succinimide ester (CFSE) labeling of mouse macrophages after Ana-1 detection mouse macrophage Ana-1 flow cytometry proliferation; 3) to detect the expression of peptide of mouse macrophage Ana-1 cell surface TLR2 and TLR4 flow cytometry.
Result
1, we used three chloroacetic acid and SDS to make peptidoglycan by boiling and trypsin digestion. After phenol sulfuric acid method, we identified two methylamino benzaldehyde and lysozyme hydrolysis. Gel filtration chromatography showed that the peptidoglycan with relatively uniform molecular weight was obtained from Lactobacillus DM9811.
2, ELISA results showed that the extracted peptidoglycan can promote mouse macrophage Ana-1 secretion of IL-10, IL-12, and peptidoglycan of low concentration can promote TNF- secretion; flow cytometry results showed that compared with the control group, peptidoglycan treated macrophages split Ana-1 generation significantly increased the number of cell surface TLR2 and TLR4 (at the same time mainly TLR4) expression were significantly increased.
conclusion
The DM9811 peptidoglycan extract of lactobacilli was shown to activate the immune activity of Ana-1 in mouse macrophages in vitro.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【引证文献】