本文選題：華美牛肝菌多糖 + 分離與純化��； 參考：《西華師范大學(xué)》2017年碩士論文

【摘要】：華美牛肝菌(Boletus speciosus Frost)隸屬牛肝菌科,牛肝菌屬,是一種可食用的大型真菌,而且具有較強(qiáng)的免疫活性和抗腫瘤的功能,是較為理想的天然食用菌。目前對華美牛肝菌的研究大多集中在培養(yǎng)馴化與栽培技術(shù)、子實(shí)體營養(yǎng)特性、多糖結(jié)構(gòu)等方面,而對華美牛肝菌多糖生物活性的研究報(bào)道較少。本課題用熱水浸提法和柱層析法對華美牛肝菌多糖進(jìn)行提取與分離純化,然后用化學(xué)法和光譜法對其結(jié)構(gòu)進(jìn)行解析,應(yīng)用現(xiàn)代生物學(xué)法研究其免疫調(diào)節(jié)以及抗腫瘤等生物活性,并以Raw264.7為例,初步研究其免疫調(diào)節(jié)活性機(jī)制。本課題通過對華美牛肝菌多糖的提取與分離純化、結(jié)構(gòu)鑒定及其生物活性的研究,為華美牛肝菌經(jīng)濟(jì)價值及藥用價值的研究開發(fā)提供理論基礎(chǔ),為進(jìn)一步大規(guī)模培養(yǎng)馴化華美牛肝菌和研究華美牛肝菌藥制品提供理論支撐和科學(xué)依據(jù),并為華美牛肝菌多糖免疫調(diào)節(jié)活性、抗腫瘤及其作用機(jī)制的研究奠定基礎(chǔ)。本文包括華美牛肝菌多糖的提取、分離與純化的研究,結(jié)構(gòu)分析鑒定,免疫調(diào)節(jié)活性與抗腫瘤活性的研究,免疫調(diào)節(jié)活性機(jī)制的初步研究四部分內(nèi)容。采用熱水浸提法和乙醇醇沉從華美牛肝菌子實(shí)體得到粗多糖,其得率為15.12%;經(jīng)DEAE-纖維素柱層析分離純化得到華美牛肝菌純多糖,并命名為BSF-X,經(jīng)過紫外全掃描發(fā)現(xiàn)不含蛋白質(zhì)和核酸,硫酸苯酚法檢測含糖量,通過高效凝膠滲透色譜法測得BSF-X的重均分子量Mw為141309 Da。通過紅外光譜分析、GC-MS分析、核磁共振分析,對多糖結(jié)構(gòu)進(jìn)行了研究。分析結(jié)果表明:華美牛肝菌多糖(BSF-X)的分子量為141309 Da,由β-D-葡萄糖和α-D-半乳糖以2:1的比例組成,具有(1→4)-β-D-葡萄糖的主鏈,6-O上連接一個→1,6)-α-D-半乳糖的側(cè)鏈,側(cè)鏈的半乳糖2-O上連接一個→4)-β-D-葡萄糖。通過CCK-8試劑盒檢測BSF-X刺激后三種免疫細(xì)胞(B淋巴細(xì)胞、T淋巴細(xì)胞、巨噬細(xì)胞)的增殖情況,周期試劑盒檢測三種細(xì)胞的細(xì)胞周期情況,中性紅法檢測巨噬細(xì)胞吞噬能力,ELISA試劑盒法檢測巨噬細(xì)胞釋放IL-6,IL-23,TNF-α的能力,發(fā)現(xiàn):BSF-X在一定濃度范圍內(nèi)能促進(jìn)三種免疫細(xì)胞的增殖,對它們處于S期的細(xì)胞數(shù)量有明顯的提高的影響;能明顯促進(jìn)Raw264.7細(xì)胞吞噬中性紅的能力;也可以促進(jìn)巨噬細(xì)胞釋放免疫因子IL-6,IL-23,TNF-α的能力。采用CCK-8法研究BSF-X體外對L929細(xì)胞的生長影響,結(jié)果表明BSF-X在一定范圍內(nèi)能夠抑制L929癌細(xì)胞的生長,且有明顯的劑量依賴關(guān)系;通過小鼠S180腫瘤模型實(shí)驗(yàn)分析,BSF-X能在體內(nèi)抑制S180腫瘤的生長,抑癌率可達(dá)61.35%。這些實(shí)驗(yàn)表明,BSF-X具有較強(qiáng)的免疫調(diào)節(jié)活性和抗腫瘤活性。通過對免疫細(xì)胞的增殖活性、巨噬細(xì)胞吞噬活性及周期研究,發(fā)現(xiàn)華美牛肝菌多糖(BSF-1)可以促進(jìn)B細(xì)胞,T細(xì)胞和巨噬細(xì)胞的增殖,通過縮短G0/G1期的細(xì)胞周期來促進(jìn)巨噬細(xì)胞的增殖和促進(jìn)S期和G2/M期的細(xì)胞周期,這可能誘導(dǎo)細(xì)胞分裂。以巨噬細(xì)胞Raw264.7為例,經(jīng)BSF-1刺激后,提取總RNA,并對轉(zhuǎn)錄組進(jìn)行差異基因分析、GO注釋和KEGG/GO通路分析,發(fā)現(xiàn)有22.22%DEG是屬于MAPK信號通路的。在MAPK信號通路中,BSF-1能通過刺激EGF與細(xì)胞表面上的表皮生長因子受體(EGFR)結(jié)合而發(fā)揮作用,它能夠?qū)е录?xì)胞增殖,分化和存活。該過程也提高了細(xì)胞內(nèi)鈣離子水平,糖酵解和蛋白質(zhì)合成,這些變化最終導(dǎo)致細(xì)胞增殖和DNA合成。該過程可充分的解釋華美牛肝菌多糖對巨噬細(xì)胞增殖活性的機(jī)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.
【學(xué)位授予單位】：西華師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S646;R282.71

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本文編號：1927446