本文選題：干酪乳桿菌LC2W + RAW264.7�。� 參考：《上海海洋大學(xué)》2010年碩士論文

【摘要】：免疫調(diào)節(jié)能力作為篩選益生菌的標(biāo)準(zhǔn)和益生菌的重要功能,近些年受到了廣泛的關(guān)注。益生菌免疫方面的研究日趨系統(tǒng)化,內(nèi)容涵蓋免疫學(xué)的各個部分,研究人員致力于益生菌治療腸道菌群失調(diào)、腫瘤、感染等方面的研究,且取得了一定的成果。益生菌制品也作為保健行業(yè)的一個新成員有著廣闊的發(fā)展空間。乳桿菌是腸道土著菌群的重要成員之一,能在腸道中定植,能改善或調(diào)節(jié)腸道微生物菌群的平衡、增強機體免疫力及抑制腫瘤的形成等。 近年來國內(nèi)外學(xué)者認為乳酸桿菌免疫調(diào)節(jié)作用與其細胞壁組分有關(guān)。本文以干酪乳桿菌LC2W為主,通過體外細胞實驗,對其細胞裂解物(cell lysate)、細胞壁(cell wall)、細胞質(zhì)(cytoplasm)以及胞外多糖(EPS)的免疫活性進行了研究。 以MRS基礎(chǔ)培養(yǎng)基對干酪乳桿菌進行增菌培養(yǎng),收集對數(shù)期菌體,洗滌菌體并采用高壓均質(zhì)破碎菌體細胞,獲得細胞裂解物(cell lysate)。將LC2W細胞裂解物經(jīng)SDS加酶處理,去蛋白、DNA及RNA,獲得純化的細胞壁(cell wall)。離心收集上清,獲得細胞質(zhì)(cytoplasm)。 巨噬細胞作為先天性免疫的重要成員,對巨噬細胞的激活與刺激作用是誘導(dǎo)宿主免疫應(yīng)答的第一步,只有激活的巨噬細胞才能發(fā)揮其免疫功能。實驗選擇觀察其受刺激后RAW264.7巨噬細胞形態(tài)、MTT代謝水平變化,研究其對巨噬細胞激活途徑的影響,結(jié)果表明,受刺激后的RAW264.7巨噬細胞形態(tài)發(fā)生明顯變化,在10μg/ml-80μg/ml濃度范圍內(nèi),對巨噬細胞RAW264.7無明顯的損傷作用,并不抑制巨噬細胞RAW264. 7增殖,不影響細胞正常的代謝能力與活性。 其次,經(jīng)10-80μg/mL的干酪乳桿菌細胞裂解物(cell lysate)、干酪乳桿菌LC2W細胞壁(cell wall)和細胞質(zhì)(cytoplasm)刺激后,巨噬細胞RAW264.7產(chǎn)生的TNF-α、IFN-γ、IL-12顯著增加,并呈一定的濃度依賴性。相同濃度下,干酪乳桿菌LC2W、WPC-200、BD-Ⅱ和Shirota四株菌之間對細胞因子的影響差異不明顯;細胞壁(cell wall)誘導(dǎo)細胞因子分泌的能力強于細胞質(zhì)(cytoplasm)。 最后,檢測了LC2W胞外多糖(EPS)對脂多糖誘導(dǎo)巨噬細胞分泌功能的影響,發(fā)現(xiàn)不論是EPS和E.coli脂多糖(LPS)同時加入或是先后間隔24h加入生長良好的RAW264.7細胞中,均能抑制由脂多糖(LPS)誘導(dǎo)的小鼠RAW264.7細胞釋放炎癥因子TNF-α,IFN-γ和IL-12,同時還能夠提高IL-10的分泌。表明LC2W胞外多糖(EPS)具有潛在的抗炎作用。
[Abstract]:As the standard of screening probiotics and the important function of probiotics, immunomodulation ability has been paid more and more attention in recent years. The study of probiotic immunity is becoming more and more systematic, covering all parts of immunology. The researchers devote themselves to the treatment of intestinal dysbacteriosis, tumor, infection and so on by probiotics, and have achieved certain results. Probiotic products as a new member of the health care industry has a broad space for development. Lactobacillus is one of the most important members of intestinal indigenous flora. It can colonize the intestinal tract, improve or regulate the balance of intestinal microbial flora, enhance immunity and inhibit tumor formation. In recent years, domestic and foreign scholars believe that the immune regulation of Lactobacillus is related to its cell wall components. In this paper, the immunological activities of Lactobacillus casei LC2W in cell lysate, cell wall, cytoplasm and extracellular polysaccharide (EPSs) were studied in vitro. Lactobacillus casei was cultured in Mrs medium. The bacteria were collected in logarithmic phase, then washed and broken by high pressure homogenization. Cell lysate was obtained. The LC2W cell lysate was treated with SDS, then the DNA and RNAs were removed, and the purified cell wall was obtained. The supernatant was collected by centrifugation and cytoplasm was obtained. As an important member of innate immunity, the activation and stimulation of macrophages is the first step to induce host immune response. Only activated macrophages can play their immune function. The effects of RAW264.7 macrophages on the activation pathway of RAW264.7 macrophages were studied. The results showed that the morphology of RAW264.7 macrophages changed significantly in the concentration range of 10 渭 g/ml-80 渭 g/ml. RAW264.7 had no obvious damage to macrophage RAW264.It did not inhibit RAW264. 7 Proliferation does not affect the normal metabolic ability and activity of cells. Secondly, stimulated by 10-80 渭 g / mL Lactobacterium casei cell lysate, Lactobacillus casei LC2W cell wall and cytoplasm, TNF- 偽 IFN- 緯 IL-12 produced by macrophage RAW264.7 was significantly increased in a dose-dependent manner. At the same concentration, there was no significant difference between the four strains of Lactobacillus casei LC2WPC-200 BD- 鈪，

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