本文選題：乳酸菌 + 胞外多糖�。� 參考：《內(nèi)蒙古大學(xué)》2017年碩士論文

【摘要】：乳酸菌胞外多糖(Lactic acid bacteria exopolysaccharide,LAB EPS)具有多種生物活性,對其研究與開發(fā)具有重要的實際意義與良好的應(yīng)用前景。本研究以實驗室保存鑒定的乳酸菌為出發(fā)菌株,篩選能夠增強細(xì)胞免疫活性的乳酸菌EPS,隨后將其進(jìn)行分離純化及結(jié)構(gòu)初步鑒定,在此基礎(chǔ)上探討其對免疫細(xì)胞活性及機體特異性免疫應(yīng)答的影響。結(jié)果如下:1.對實驗室保存鑒定的110株乳酸桿菌所產(chǎn)EPS進(jìn)行測定,篩選10株高產(chǎn)EPS 乳酸桿菌,其中Lactobacillus harbinensis TCP086、Lactobaillus kefiri SXJ29和Lactobacillus casei SXJ30所產(chǎn)EPS可以增強巨噬細(xì)胞增殖、吞噬以及NO釋放能力。2.將上述 Lactobacillus harbinensis TCP086、Lactobacillus kefiri SXJ29 和Lactobacillus casei SXJ30 所產(chǎn) EPS 經(jīng)過 DEAE-Sepharose Fast Flow 和 Sepharose CL-6B色譜柱進(jìn)行分離純化后獲得主要均一組分:TCP086 EPS、SXJ29 EPS和SXJ30 EPS。紫外光譜和紅外光譜掃描發(fā)現(xiàn)三種EPS均不含核酸和蛋白質(zhì),且均具有多糖的特征吸收峰,存在αα型吡喃糖環(huán)構(gòu)型;多角度激光光散射儀檢測其分子量分別為4.908 × 104 Da、3.423 × 104 Da和3.737 × 104 Da;離子色譜檢測單糖組成發(fā)現(xiàn),三種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種單糖組成,其中TCP086 EPS各主要組分摩爾比氨基葡萄糖:氨基半乳糖:甘露糖:葡萄糖醛酸約為2.6:1.6:1.2:1;SXJ29EPS各主要組分摩爾比葡萄糖:氨基半乳糖:氨基葡萄糖約為3.1:1:1;SXJ30 EPS各主要組分摩爾比葡萄糖:氨基葡萄糖:甘露糖約為1.4:1.1:1。3.進(jìn)一步以純化的TCP086 EPS、SXJ29 EPS和SXJ30 EPS刺激巨噬細(xì)胞,對其增殖能力、吞噬中性紅能力、NO釋放能力、細(xì)胞因子分泌以及表面分子CD40、CD80、CD86和MHC-Ⅱ表達(dá)進(jìn)行檢測后發(fā)現(xiàn),巨噬細(xì)胞的免疫增強活性與EPS的濃度呈正相關(guān),且SXJ30 EPS對巨噬細(xì)胞的免疫增強活性最強。4.在體外成功利用IL-4和GM-CSF誘導(dǎo)獲得小鼠骨髓來源樹突狀細(xì)胞,顯微鏡下觀察SXJ30EPS刺激小鼠BMDCs后,細(xì)胞突起增多,體積增大;流式細(xì)胞術(shù)和ELISA法檢測后發(fā)現(xiàn),SXJ30 EPS能顯著增強小鼠BMDCs表面CD40、CD80、CD86和MHC-Ⅱ類分子的表達(dá)以及細(xì)胞因子(TNF-α、IL-6、IL-10和IL-12)的分泌。5.將SXJ30 EPS與OVA抗原免疫小鼠,發(fā)現(xiàn)SXJ30 EPS可以顯著提高小鼠脾臟DCs表面CD40、CD80、CD86和MHC-Ⅱ類分子的表達(dá)量;與鋁鹽(Alum)佐劑相比,SXJ30 EPS可以顯著提高小鼠血清中OVA特異性IgG抗體以及IgG抗體亞類IgG1、IgG2a和IgG2b的滴度;此外,SXJ30EPS還能顯著促進(jìn)脾臟T淋巴細(xì)胞增殖以及INF-γ和IL-4的分泌;顯著提高脾臟IL-4+ CD4+ T細(xì)胞、IFN-γ+CD4+ T細(xì)胞以及IFN-γ+ CD8+ T細(xì)胞的比例,與Alum佐劑相比差異顯著。上述結(jié)果提示:SXJ30 EPS在體外不僅可以增強巨噬細(xì)胞免疫活性,還能促進(jìn)樹突狀細(xì)胞成熟和前炎性因子分泌。SXJ30 EPS可以顯著提高小鼠對OVA抗原Th1型和Th2型免疫應(yīng)答,其中主要以Th2型免疫應(yīng)答為主。該研究為免疫增強劑的篩選和應(yīng)用提供了可靠的理論指導(dǎo)和實驗資料。
[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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 賀淼;黃鑫;李彪;胡駿鵬;;酵母細(xì)胞壁多糖對肉雞生長性能、免疫器官發(fā)育及盲腸微生物的影響[J];飼料工業(yè);2015年10期

2 劉鷺;潘道東;丁琳;曾小群;;硒化乳酸菌胞外多糖對小鼠腹腔巨噬細(xì)胞及腫瘤細(xì)胞內(nèi)游離Ca~(2+)的影響[J];食品科學(xué);2014年01期

3 羅燕;邵永斌;谷新利;李星艷;;淫羊藿多糖對雞免疫功能及疫苗免疫效果的影響[J];中國家禽;2009年01期

4 王瑞瓊;張紅星;熊利霞;易欣欣;劉慧;;乳酸菌胞外多糖分離純化方法研究進(jìn)展[J];食品科學(xué);2008年08期

5 李康;郭強;王翠妮;陳敏;徐薇;熊思東;;M1和M2型巨噬細(xì)胞表型的比較分析[J];現(xiàn)代免疫學(xué);2008年03期

6 王文平;郭祀遠(yuǎn);李琳;王明力;梁桂娟;;苯酚-硫酸法測定野木瓜中多糖含量的研究[J];食品科學(xué);2007年04期

7 ;Exopolysaccharides from Marine Bacteria[J];Journal of Ocean University of China;2005年01期

8 趙玉臣,王迎俊,王忠鳳;乳酸菌胞外多糖[J];中國乳業(yè);2003年04期

9 鐘鳴,張德山,Murata J,Saiki I,山本格;距骨多糖 (APS)和距骨類黃酮 (AF)對活體外正常老鼠細(xì)胞和瘤變老鼠細(xì)胞的淋巴細(xì)胞繁殖及T-淋巴細(xì)胞亞群的影響(英文)[J];四川大學(xué)學(xué)報(自然科學(xué)版);2001年06期

10 石榴;方怡;袁偉鋒;李理;黃文杰;;脂多糖刺激RAW264.7和Ana-1形態(tài)改變及細(xì)胞因子表達(dá)的差異[J];國際呼吸雜志;2009年13期

相關(guān)博士學(xué)位論文 前2條

1 馮美琴;植物乳桿菌胞外多糖發(fā)酵、結(jié)構(gòu)鑒定及其功能特性研究[D];南京農(nóng)業(yè)大學(xué);2012年

2 黃丹菲;植物活性多糖對小鼠樹突狀細(xì)胞和巨噬細(xì)胞功能的調(diào)節(jié)作用[D];南昌大學(xué);2009年

，

本文編號：1986129