干酪乳杆菌LC2W细胞组分对巨噬细胞活性及细胞因子分泌的影响

发布时间：2018-06-17 14:41

  本文选题：干酪乳杆菌LC2W + RAW264.7　； 参考：《上海海洋大学》2010年硕士论文

【摘要】：免疫调节能力作为筛选益生菌的标准和益生菌的重要功能,近些年受到了广泛的关注。益生菌免疫方面的研究日趋系统化,内容涵盖免疫学的各个部分,研究人员致力于益生菌治疗肠道菌群失调、肿瘤、感染等方面的研究,且取得了一定的成果。益生菌制品也作为保健行业的一个新成员有着广阔的发展空间。乳杆菌是肠道土著菌群的重要成员之一,能在肠道中定植,能改善或调节肠道微生物菌群的平衡、增强机体免疫力及抑制肿瘤的形成等。 近年来国内外学者认为乳酸杆菌免疫调节作用与其细胞壁组分有关。本文以干酪乳杆菌LC2W为主,通过体外细胞实验,对其细胞裂解物(cell lysate)、细胞壁(cell wall)、细胞质(cytoplasm)以及胞外多糖(EPS)的免疫活性进行了研究。 以MRS基础培养基对干酪乳杆菌进行增菌培养,收集对数期菌体,洗涤菌体并采用高压均质破碎菌体细胞,获得细胞裂解物(cell lysate)。将LC2W细胞裂解物经SDS加酶处理,去蛋白、DNA及RNA,获得纯化的细胞壁(cell wall)。离心收集上清,获得细胞质(cytoplasm)。 巨噬细胞作为先天性免疫的重要成员,对巨噬细胞的激活与刺激作用是诱导宿主免疫应答的第一步,只有激活的巨噬细胞才能发挥其免疫功能。实验选择观察其受刺激后RAW264.7巨噬细胞形态、MTT代谢水平变化,研究其对巨噬细胞激活途径的影响,结果表明,受刺激后的RAW264.7巨噬细胞形态发生明显变化,在10μg/ml-80μg/ml浓度范围内,对巨噬细胞RAW264.7无明显的损伤作用,并不抑制巨噬细胞RAW264. 7增殖,不影响细胞正常的代谢能力与活性。 其次,经10-80μg/mL的干酪乳杆菌细胞裂解物(cell lysate)、干酪乳杆菌LC2W细胞壁(cell wall)和细胞质(cytoplasm)刺激后,巨噬细胞RAW264.7产生的TNF-α、IFN-γ、IL-12显著增加,并呈一定的浓度依赖性。相同浓度下,干酪乳杆菌LC2W、WPC-200、BD-Ⅱ和Shirota四株菌之间对细胞因子的影响差异不明显;细胞壁(cell wall)诱导细胞因子分泌的能力强于细胞质(cytoplasm)。 最后,检测了LC2W胞外多糖(EPS)对脂多糖诱导巨噬细胞分泌功能的影响,发现不论是EPS和E.coli脂多糖(LPS)同时加入或是先后间隔24h加入生长良好的RAW264.7细胞中,均能抑制由脂多糖(LPS)诱导的小鼠RAW264.7细胞释放炎症因子TNF-α,IFN-γ和IL-12,同时还能够提高IL-10的分泌。表明LC2W胞外多糖(EPS)具有潜在的抗炎作用。
[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- 伪 IFN- 纬 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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