本文關(guān)鍵詞： 乳酸桿菌 肽聚糖 提取 免疫活性　出處：《大連醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：背景 乳酸桿菌是人體中一種非常重要的益生菌,其生物學(xué)作用包括生物拮抗,營養(yǎng)作用,免疫調(diào)節(jié),抗腫瘤,抗衰老等作用。乳酸桿菌通過其特異的細(xì)胞組分與宿主的免疫細(xì)胞相互作用而發(fā)揮免疫調(diào)節(jié)功能。 肽聚糖(peptidoglycan)也稱黏肽(mucopeptide),是乳酸桿菌細(xì)胞壁結(jié)構(gòu)和理化性質(zhì)的主要功能性物質(zhì)。肽聚糖的基本結(jié)構(gòu)是由N-乙酰葡萄糖(GlcNAc)和N-乙酰胞壁酸(MurNAc)通過β-1,4糖苷鍵連接,每個N-乙酰胞壁酸(MurNAc)又連接一個四肽側(cè)鏈,每個四肽側(cè)鏈又通過一個五聚甘氨酸組成的肽交聯(lián)橋(peptide across bridge)連接,肽聚糖是一類由共價鍵連接,包圍整個細(xì)菌細(xì)胞壁的囊狀大分子(sac-liked macromolecule)。近年來,肽聚糖的生物活性一直受到關(guān)注。 Toll樣受體(Toll like receptor,TLR)屬于膜識別受體,是近年發(fā)現(xiàn)的一種膜表面受體。TLR樣受體通過上調(diào)抗原遞呈細(xì)胞表面的共刺激分子及抗原遞呈細(xì)胞分泌的細(xì)胞因子,誘導(dǎo)T和B淋巴細(xì)胞向效應(yīng)T和B淋巴細(xì)胞分化,進(jìn)而調(diào)節(jié)獲得性免疫。TLRs可以識別微生物所特有的保守結(jié)構(gòu),這類保守結(jié)構(gòu)稱為微生物相關(guān)保守分子(pathogen associated molecular pattern,PAMP)。 肽聚糖是乳酸桿菌含量最豐富的MAMP之一,可能通過TLRs的識別而發(fā)揮免疫作用。 目的 本文以大連醫(yī)科大學(xué)微生物教研室分離的一株乳酸桿菌DM9811為研究對象,通過提取其細(xì)胞壁的主要成分肽聚糖,觀察肽聚糖對小鼠巨噬細(xì)胞Ana-1的免疫調(diào)節(jié)作用,為進(jìn)而探索肽聚糖發(fā)揮免疫調(diào)節(jié)作用的分子機(jī)制提供實(shí)驗(yàn)基礎(chǔ)。 方法 1、肽聚糖的分離純化 收集培養(yǎng)48小時的乳酸桿菌DM9811菌體,經(jīng)10%三氯乙酸煮沸20分鐘和含有4%SDS的PBS煮沸30分鐘后,再用胰酶消化48小時,離心收集的沉淀即為肽聚糖粗提取物。肽聚糖粗提取物進(jìn)一步溶菌酶酶解后,經(jīng)葡聚糖凝膠SephacrylS-300HR進(jìn)行分離和部分純化。 2、肽聚糖鑒定 應(yīng)用苯酚-硫酸法測定總糖含量,對二甲氨基苯甲醛顯色聯(lián)合溶菌酶水解實(shí)驗(yàn)測定N-乙酰葡萄糖,測定酶解過程中溶液吸光度的變化情況來鑒定肽聚糖。 3、肽聚糖的免疫活性 肽聚糖處理小鼠巨噬細(xì)胞Ana-1前后,分別1)應(yīng)用酶聯(lián)免疫吸附劑測定法(enzyme linked immunosorbent assay, ELISA)檢測培養(yǎng)上清中的IL-10,IL-12和TNF-α的蛋白水平;2)在活細(xì)胞染料羧基熒光素乙酰乙酸琥珀酸亞胺酯(CFSE)標(biāo)記小鼠巨噬細(xì)胞Ana-1后,流式細(xì)胞術(shù)檢測小鼠巨噬細(xì)胞Ana-1的增殖分裂;3)流式細(xì)胞術(shù)檢測肽小鼠巨噬細(xì)胞Ana-1細(xì)胞表面TLR2和TLR4的表達(dá)。 結(jié)果 1、應(yīng)用三氯乙酸和SDS煮沸以及胰酶酶解得到肽聚糖,經(jīng)苯酚-硫酸法,對二甲氨基苯甲醛顯色,溶菌酶水解實(shí)驗(yàn)鑒定,凝膠過濾層析顯示從乳酸桿菌DM9811獲得了分子量相對均一的肽聚糖。 2、ELISA結(jié)果顯示所提取的肽聚糖可以促進(jìn)小鼠巨噬細(xì)胞Ana-1分泌IL-10,IL-12,而低濃度的肽聚糖可以促進(jìn)TNF-α分泌;流式細(xì)胞技術(shù)檢測結(jié)果顯示,與對照組相比,肽聚糖處理的巨噬細(xì)胞Ana-1分裂代數(shù)明顯增加,同時細(xì)胞表面TLR2和TLR4(主要是TLR4)表達(dá)均顯著提高。 結(jié)論 乳酸桿菌DM9811肽聚糖提取物表現(xiàn)在體外激活小鼠巨噬細(xì)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.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前2條

1 張玲;楊彩梅;曹廣添;肖英平;曾新福;;Toll樣受體與益生菌免疫[J];動物營養(yǎng)學(xué)報(bào);2013年01期

2 謝警鴻;邱德全;劉春曉;朱衛(wèi)衛(wèi);曾林;;溶藻弧菌肽聚糖對凡納濱對蝦蝦青素、免疫指標(biāo)及保護(hù)率的影響[J];廣東海洋大學(xué)學(xué)報(bào);2013年01期

，

本文編號：1494122