益生菌粘附及免疫调节机理的研究

发布时间：2018-06-08 16:43

本文选题：益生菌 + 粘附　；参考：《东北农业大学》2005年硕士论文

【摘要】：本试验以青春双歧杆菌和嗜酸乳杆菌为对象,对益生菌在机体肠道内的粘附定植以及提高免疫功能的机理进行了研究,目的在于寻找出益生菌产生粘附作用的物质,以及在肠道细胞上的粘附素受体,以及益生菌产生免疫促进作用成分。双歧杆菌和乳杆菌都属于革兰氏阳性菌。革兰氏阳性菌菌体细胞壁表面主要由肽聚糖(Peptidoglycan,PG)、多糖(Polysaccharide,Ps)和表层蛋白质(S-层蛋白,Surface-Layerprotein,S-layerprotein,SLP)组成,这些物质在益生菌菌体与宿主肠道细胞的粘附过程中直接或间接的起到不同程度的作用。本实验对益生菌粘附机理的研究主要集中在于菌体代谢产物和细胞表层成分,即肽聚糖和表层的蛋白质。实验中首先用TritonX-100结合胰蛋白酶、DNA酶、RNA酶和蛋白酶E的方法,对青春双歧杆菌菌体细胞壁所含的完整肽聚糖进行提取;采用十二烷基硫酸钠(sDS)结合胰蛋白酶、三氯乙酸处理的方法提取嗜酸乳杆菌菌体细胞壁所含肽聚糖,并确定了最适的提取条件为用质量浓度为80g/L的十二烷基硫酸钠,沸水浴处理10min,然后用胰蛋白酶和三氯乙酸连续处理;同时对提取物进行蛋白质含量的测定和氨基酸分析,以确定提取过程是否真正有效的去除了受试菌株菌体内含有的蛋白质。通过不同方法分别对受试菌株代谢产物、菌体表面成分以及肠道细胞表面物质在粘附过程中的作用进行了研究。确定青春双歧杆菌的粘附素为菌体分泌到培养基中的一种蛋白质,肠道上皮细胞对青春双歧的粘附素受体为细胞表面的一种糖蛋白;嗜酸乳杆菌的粘附素是菌体细胞壁上的一种蛋白质,其在肠道上皮细胞上的粘附受体为蛋白。实验中还对灭活青春双歧杆菌和嗜酸乳杆菌菌株的粘附能力进行了初步研究,发现经热处理失活后的青春双歧杆菌依然具有粘附能力,而且并未受到明显影响;而嗜酸乳杆菌在经过热处理后,则基本丧失粘附能力。提高机体免疫力是益生菌的主要功效之一。本实验以昆明小鼠为对象进行动物实验,检测青春双歧杆菌与嗜酸乳杆菌细胞壁肽聚糖促进免疫功能的作用。结果显示,与对照样本相比,接受菌液注射和肽聚糖注射都会对受试动物的免疫指标产生影响,其血清中自介素IL-6水平、血清溶菌酶活力、腹腔巨噬细胞数量、巨噬细胞吞噬能力都得到明显提高,说明细胞壁肽聚糖对机体免疫功能具有明显促进作用。
[Abstract]:In this study, Bifidobacterium pubertii and Lactobacillus acidophilus were used to study the adhesion and colonization of probiotics in the intestinal tract and the mechanism of improving immune function. Bifidobacterium and Lactobacillus belong to Gram-positive bacteria. The cell wall surface of Gram-positive bacteria is mainly composed of peptidoglycanine (PGN), polysaccharide (PS) and surface-Layer protein (SLP). These substances directly or indirectly play an important role in the adhesion of probiotic bacteria to intestinal cells. In this study, the adhesion mechanism of probiotics was mainly focused on the metabolites of probiotics and the surface components of the cells. That is, peptidoglycan and surface protein. In the experiment, the intact peptidoglycan contained in cell wall of bifidobacterium pubescens was extracted by Triton X-100 binding trypsin DNA enzyme and protease E. Sodium dodecyl sulfate combined with trypsin was used to extract peptidoglycan from the cell wall of Bifidobacterium pubescens. The peptidoglycan from the cell wall of Lactobacillus acidophilus was extracted by trichloroacetic acid, and the optimum extraction conditions were determined as follows: sodium dodecyl sulfate, 80 g / L mass concentration, The extract was treated with trypsin and trichloroacetic acid for 10 min, and the protein content and amino acid were determined. In order to determine whether the extraction process is really effective in removing the protein contained in the tested strain, the metabolites of the tested strain were obtained by different methods. The effects of bacterial surface composition and intestinal cell surface material on adhesion were studied. The adhesion factor of bifidobacterium pubertidis was identified as a protein secreted into the culture medium, and the adhesin receptor of intestinal epithelial cells to the bifidobacterium was identified as a glycoprotein on the cell surface. The adhesion hormone of Lactobacillus acidophilus is a protein in the cell wall of Lactobacillus acidophilus. Its adhesion receptor on intestinal epithelial cells was protein. The adhesion ability of inactivated bifidobacterium and Lactobacillus acidophilus strains were also studied. It was found that Bifidobacterium pubertifolia after heat treatment still had adhesion ability and was not significantly affected, while Lactobacillus acidophilus was not affected after heat treatment. Improving immunity is one of the main effects of probiotics. The effects of peptidoglycan on cell wall of Bifidobacterium puberty and Lactobacillus acidophilus were studied in Kunming mice. The results showed that, compared with the control sample, inoculation of bacterial fluid and peptidoglycan had an effect on the immune indexes of the tested animals. The serum levels of IL-6, lysozyme activity and the number of peritoneal macrophages were determined. Phagocytosis of macrophages was improved obviously, which indicated that peptidoglycan could promote immune function.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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