华美牛肝菌多糖（BSF-X）的结构鉴定、生物活性及华美牛肝菌多糖（BSF-1）免疫活性机制的研究

发布时间：2018-05-24 03:07

本文选题：华美牛肝菌多糖 + 分离与纯化　；参考：《西华师范大学》2017年硕士论文

【摘要】：华美牛肝菌(Boletus speciosus Frost)隶属牛肝菌科,牛肝菌属,是一种可食用的大型真菌,而且具有较强的免疫活性和抗肿瘤的功能,是较为理想的天然食用菌。目前对华美牛肝菌的研究大多集中在培养驯化与栽培技术、子实体营养特性、多糖结构等方面,而对华美牛肝菌多糖生物活性的研究报道较少。本课题用热水浸提法和柱层析法对华美牛肝菌多糖进行提取与分离纯化,然后用化学法和光谱法对其结构进行解析,应用现代生物学法研究其免疫调节以及抗肿瘤等生物活性,并以Raw264.7为例,初步研究其免疫调节活性机制。本课题通过对华美牛肝菌多糖的提取与分离纯化、结构鉴定及其生物活性的研究,为华美牛肝菌经济价值及药用价值的研究开发提供理论基础,为进一步大规模培养驯化华美牛肝菌和研究华美牛肝菌药制品提供理论支撑和科学依据,并为华美牛肝菌多糖免疫调节活性、抗肿瘤及其作用机制的研究奠定基础。本文包括华美牛肝菌多糖的提取、分离与纯化的研究,结构分析鉴定,免疫调节活性与抗肿瘤活性的研究,免疫调节活性机制的初步研究四部分内容。采用热水浸提法和乙醇醇沉从华美牛肝菌子实体得到粗多糖,其得率为15.12%;经DEAE-纤维素柱层析分离纯化得到华美牛肝菌纯多糖,并命名为BSF-X,经过紫外全扫描发现不含蛋白质和核酸,硫酸苯酚法检测含糖量,通过高效凝胶渗透色谱法测得BSF-X的重均分子量Mw为141309 Da。通过红外光谱分析、GC-MS分析、核磁共振分析,对多糖结构进行了研究。分析结果表明:华美牛肝菌多糖(BSF-X)的分子量为141309 Da,由β-D-葡萄糖和α-D-半乳糖以2:1的比例组成,具有(1→4)-β-D-葡萄糖的主链,6-O上连接一个→1,6)-α-D-半乳糖的侧链,侧链的半乳糖2-O上连接一个→4)-β-D-葡萄糖。通过CCK-8试剂盒检测BSF-X刺激后三种免疫细胞(B淋巴细胞、T淋巴细胞、巨噬细胞)的增殖情况,周期试剂盒检测三种细胞的细胞周期情况,中性红法检测巨噬细胞吞噬能力,ELISA试剂盒法检测巨噬细胞释放IL-6,IL-23,TNF-α的能力,发现:BSF-X在一定浓度范围内能促进三种免疫细胞的增殖,对它们处于S期的细胞数量有明显的提高的影响;能明显促进Raw264.7细胞吞噬中性红的能力;也可以促进巨噬细胞释放免疫因子IL-6,IL-23,TNF-α的能力。采用CCK-8法研究BSF-X体外对L929细胞的生长影响,结果表明BSF-X在一定范围内能够抑制L929癌细胞的生长,且有明显的剂量依赖关系;通过小鼠S180肿瘤模型实验分析,BSF-X能在体内抑制S180肿瘤的生长,抑癌率可达61.35%。这些实验表明,BSF-X具有较强的免疫调节活性和抗肿瘤活性。通过对免疫细胞的增殖活性、巨噬细胞吞噬活性及周期研究,发现华美牛肝菌多糖(BSF-1)可以促进B细胞,T细胞和巨噬细胞的增殖,通过缩短G0/G1期的细胞周期来促进巨噬细胞的增殖和促进S期和G2/M期的细胞周期,这可能诱导细胞分裂。以巨噬细胞Raw264.7为例,经BSF-1刺激后,提取总RNA,并对转录组进行差异基因分析、GO注释和KEGG/GO通路分析,发现有22.22%DEG是属于MAPK信号通路的。在MAPK信号通路中,BSF-1能通过刺激EGF与细胞表面上的表皮生长因子受体(EGFR)结合而发挥作用,它能够导致细胞增殖,分化和存活。该过程也提高了细胞内钙离子水平,糖酵解和蛋白质合成,这些变化最终导致细胞增殖和DNA合成。该过程可充分的解释华美牛肝菌多糖对巨噬细胞增殖活性的机制。
[Abstract]:Boletus speciosus Frost, which belongs to the family of Boletus and Boletus, is a large edible fungus, and has strong immune activity and anti-tumor function. It is an ideal natural edible fungus. At present, most of the studies on Boletus Huamei mostly focus on the cultivation and cultivation techniques, the nutrient characteristics of the fruiting bodies, and the polysaccharides. There are few reports on the biological activity of Boletus polysaccharides. This subject uses hot water extraction and column chromatography to extract and separate the polysaccharide of Boletus Boletus, and then analyses its structure by chemical and spectral methods, and studies its immunomodulatory and anti-tumor biological activity by modern biological method. The mechanism of immunomodulatory activity was preliminarily studied with Raw264.7 as an example. The study provided a theoretical basis for the research and development of the economic value and medicinal value of Boletus by extracting and purifying the polysaccharides of Boletus Boletus, and the research and development of its biological activity. It provides theoretical support and scientific basis for the study of Boletus, and lays the foundation for the study of the immunoregulation activity of Polysaccharide from Boletus Boletus, anti-tumor and its mechanism. This article includes the study on the extraction, isolation and purification of Boletus polysaccharide, structural analysis and identification, immunomodulating activity and anti-tumor activity, and immunization. The four part of the preliminary study on the mechanism of regulating activity. The crude polysaccharide was obtained by hot water extraction and ethanol alcohol precipitation from the fruiting body of Huamei liver. The yield was 15.12%. The pure polysaccharides of Boletus Huamei were separated and purified by DEAE- cellulose column chromatography, and named BSF-X, and the protein and nucleic acid, phenol sulphate were found by UV full scanning. The content of sugar was detected by the method. The weight average molecular weight of BSF-X was measured by high performance gel permeation chromatography (Mw) to 141309 Da.. The structure of polysaccharide was studied by infrared spectroscopy, GC-MS analysis and nuclear magnetic resonance analysis. The results showed that the molecular weight of Boletus polysaccharide (BSF-X) was 141309 Da, from beta -D- glucose and alpha -D- galactose to 2:1. Proportions, with the main chain of (1 to 4) - beta -D- glucose, the side chain of a - 1,6 - alpha - -D- - -D- semi lactose, and a - 4 - beta -D- glucose on the side chain galactose 2-O. The proliferation of three immune cells (B lymphocyte, T nelba cell, macrophage) after BSF-X stimulation was detected by CCK-8 kit, and three of the periodic kit was detected. The cell cycle of the species, the neutral red method was used to detect the phagocytosis of macrophages. The ELISA kit method was used to detect the ability of macrophages to release IL-6, IL-23 and TNF- alpha. It was found that BSF-X could promote the proliferation of three immune cells in a certain concentration range, and obviously improve the number of cells in the S phase, and can obviously promote Raw264.. The ability of 7 cells to phagocyt neutral red and to promote the ability of macrophages to release immune factors IL-6, IL-23, TNF- alpha. The effect of BSF-X on the growth of L929 cells in vitro was studied by CCK-8 method. The results showed that BSF-X could inhibit the growth of L929 cancer cells in a certain range, and there was a clear dose dependence; the S180 tumor model in mice was real. BSF-X can inhibit the growth of S180 tumor in the body, and the tumor suppressor rate can reach 61.35%.. These experiments show that BSF-X has strong immunomodulatory activity and anti-tumor activity. Through the study of the proliferation activity of immune cells, phagocytic activity and cycle of macrophages, it is found that BSF-1 can promote B cells, T cells and macrophages. Cell proliferation, by shortening the cell cycle of the G0/G1 phase to promote the proliferation of macrophages and promote the cell cycle of phase S and G2/M phase, which may induce cell division. Taking macrophage Raw264.7 as an example, after BSF-1 stimulation, the total RNA is extracted, and the differential gene analysis of the transcriptional group, GO annotation and KEGG/GO pathway analysis have been found, and 22.22%DEG is found to be 22.22%DEG It belongs to the MAPK signaling pathway. In the MAPK signaling pathway, BSF-1 can play a role by stimulating the binding of EGF to the epidermal growth factor receptor (EGFR) on the cell surface, which can lead to cell proliferation, differentiation and survival. This process also improves intracellular calcium levels, glycolysis and protein synthesis, and these changes eventually lead to cell growth. Colonization and DNA synthesis, which can fully explain the mechanism of Boletus polysaccharides on macrophage proliferation.
【学位授予单位】：西华师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S646;R282.71

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