乳酸杆菌对LPS致敏小肠上皮细胞的免疫调节效应及机制研究

发布时间：2018-06-23 15:33

本文选题：乳酸杆菌 + LGG　；参考：《浙江农林大学》2015年硕士论文

【摘要】：益生菌广泛用于调节动物肠道健康,乳酸杆菌为应用最早、研究最多的益生菌菌种之一。乳酸杆菌通过其不同成分能够介导动物胃肠道免疫调节作用,发挥益生效应,然而其免疫调节机制仍不明了。鉴于此,阐明乳酸杆菌具有怎么样的肠道免疫调节功能,解析其免疫调节的信号通路及其关键靶点,将为益生菌免疫调节功能的开发提供科学依据,对筛选和培育高效乳酸杆菌等益生菌具有重要的实践意义。本试验旨在以公认的标准益生乳酸杆菌——鼠李糖乳酸杆菌(Lactobacillus rhamnosus GG,LGG)为代表,同时分离并获得LGG不同成分(表面蛋白SLP、胞外多糖EPS、基因组DNA以及人工合成的未甲基化CpG ODN),分析LGG及其不同成分对人工致敏体外培养肠上皮细胞的免疫调节效应和免疫保护作用,查明乳酸杆菌对小肠上皮细胞信号通路的调控作用,解析乳酸杆菌实现免疫调节作用的信号通路靶点,阐明乳酸杆菌发挥肠道免疫调控作用的分子机制。与此同时,以LGG作为参考菌株,构建小鼠炎症模型研究猪源乳酸杆菌(高粘附菌株Lactobacillus reuteri ZJ617和低粘附菌株Lactobacillus reuteri ZJ615)对肠道的免疫调节作用,分析乳酸杆菌介导动物肠道免疫调节作用的机制。本研究主要采用大肠杆菌LPS分别诱导猪小肠上皮细胞系IPEC-J2细胞和C57BL/6小鼠产生免疫反应,构建炎症模型以评定乳酸杆菌的免疫调节作用。通过构建猪小肠上皮细胞系IPEC-J2细胞脂多糖LPS炎症模型,采用2×107 CFU/mL LGG或从LGG中分离纯化获得的不同成分(表面蛋白SLP、基因组DNA、胞外多糖EPS以及人工合成的未甲基化CpG ODN序列),分别预处理IPEC-J2细胞(1×106细胞/孔)4小时,再以脂多糖LPS刺激细胞。同时,通过构建C57BL/6小鼠LPS炎症模型,连续灌胃108 CFU/mL乳酸杆菌(LGG、ZJ617或ZJ615)一周后,腹腔注射LPS诱发炎症。采用qRT-PCR技术检测IPEC-J2细胞和小鼠肠道组织致炎性细胞因子以及Toll样受体(TLR)的mRNA水平。采用Western-blot技术、免疫组化和免疫荧光技术分别检测IPEC-J2细胞和小鼠肠道组织中炎症相关信号通路(NF-κB信号通路和MAPK信号通路)主要靶点蛋白的表达水平。研究结果表明:1)与单纯LPS刺激猪肠上皮IPEC-J2细胞相比,LGG预处理IPEC-J2细胞后显著降低由LPS诱导的细胞致炎性细胞因子IL-6、IL-12、TNF-α和TLR受体TLR2、TLR4、TLR9的mRNA水平;细胞信号通路分子p38MAPK、ERK1/2和NF-κBp65的磷酸化水平显著降低,而I-κBα表达量显著上升(P0.05)。2)与单纯LPS刺激IPEC-J2细胞相比,LGG表面蛋白SLP和胞外多糖EPS预处理IPEC-J2细胞后能够显著降低由LPS诱导的细胞致炎性细胞因子IL-6、IL-12、TNF-α和TLR受体TLR2、TLR4、TLR9的mRNA水平;细胞信号通路分子p38MAPK和NF-κBp65磷酸化水平显著降低,而I-κBα表达量显著上升(P0.05)。3)与单纯LPS刺激IPEC-J2细胞相比,LGG未甲基化的CpG ODN预处理IPEC-J2细胞能够显著升高由LPS诱导的细胞致炎性细胞因子IL-6、IL-12、TNF-α水平和TLR受体TLR2、TLR4、TLR9的mRNA水平;信号通路分子p38MAPK、ERK1/2磷酸化水平与单纯LPS刺激细胞相比没有显著差异(P0.05)。4)与单纯LPS刺激未经乳酸杆菌灌胃小鼠相比,LGG和低粘附力乳酸杆菌ZJ615连续灌胃一周的小鼠接受LPS刺激后,其肠道组织中炎性细胞因子IL-6、IL-12、TNF-α和TLR受体TLR2、TLR4、TLR9的mRNA水平显著下降;肠道组织中信号通路分子p38MAPK、ERK1/2和NF-κBp65的磷酸化水平显著降低,而I-κBα表达量显著上升(P0.05)。5)与单纯LPS刺激未经乳酸杆菌灌胃小鼠相比,高粘附力乳酸杆菌ZJ617连续灌胃一周的小鼠接受LPS刺激后,其肠道组织中炎性细胞因子IL-6、IL-12、TNF-α和TLR受体TLR2、TLR4、TLR9的mRNA水平显著升高,同时小鼠肠道组织中抗炎性细胞因子IL-10的mRNA水平显著升高;肠道组织中信号通路分子I-κBα表达量显著下降(P0.05)。研究结论:由此可见,鼠李糖乳酸杆菌LGG主要通过调节小肠上皮细胞Toll样受体mRNA水平,抑制细胞MAPK和NF-κB信号通路激活,降低LPS诱导的炎性细胞因子mRNA水平,从而介导肠道免疫调节作用。鼠李糖乳酸杆菌LGG成分表面蛋白SLP、胞外多糖EPS以及未甲基化CpG ODN对LPS诱导的肠上皮细胞具有免疫调控作用,其中未甲基化CpG ODN具有免疫刺激作用。不同粘附力乳酸杆菌肠道免疫调节效应存在差异,其中低粘附力乳酸杆菌ZJ615介导肠道免疫调控作用与LGG相似。
[Abstract]:Probiotics are widely used to regulate the intestinal health of animals. Lactobacillus is one of the earliest and most studied probiotic bacteria. Lactobacilli can mediate the immune regulation effect of animal gastrointestinal tract through its different components and play probiotic effect. However, the immune regulation mechanism is still unknown. The signal transduction pathway and the key target of the immunoregulation will provide a scientific basis for the development of the immunoregulation function of probiotics, and it is of great practical significance for the screening and breeding of probiotics such as the efficient Lactobacillus. This experiment aims at the recognized standard probiotic Lactobacillus, Lactobacillus Rhamnus (Lactob). Acillus rhamnosus GG, LGG) as the representative, at the same time separate and obtain the different components of LGG (surface protein SLP, extracellular polysaccharide EPS, genomic DNA, and synthetic CpG ODN), analyze the immunological and immune protective effects of LGG and its different components on the cultured intestinal epithelial cells in vitro, and identify the lactobacillus to the small intestine. The regulatory role of epithelial cell signaling pathway, the signal pathway target of Lactobacillus to realize immunoregulation, and the molecular mechanism of lactobacilli to play the role of intestinal immune regulation. At the same time, LGG is used as a reference strain to construct a mouse model of lactobacilli (high adhesion strain Lactobacillus reuteri ZJ617). The immunoregulation effect of low adhesion strain Lactobacillus reuteri ZJ615 on intestinal tract was analyzed. The mechanism of lactic acid bacilli mediated intestinal immunoregulation in animals was analyzed. This study mainly used Escherichia coli LPS to induce the immune response of IPEC-J2 and C57BL/6 mice in small intestinal epithelial cell lines, and to construct an inflammatory model to assess the lactic acid lever. By constructing the inflammatory model of lipopolysaccharide LPS in the IPEC-J2 cell line of the pig small intestinal epithelial cell line, the different components (surface protein SLP, genomic DNA, extracellular polysaccharide EPS, and synthetic CpG ODN sequence) were obtained by constructing the inflammatory model of lipopolysaccharide in the IPEC-J2 cell line of the pig small intestinal epithelial cell line (1 * 106 fines, respectively, for the sequence of the surface protein SLP, the genomic DNA, the extracellular polysaccharide EPS and the synthetic CpG ODN sequence). At the same time, the cells were stimulated by lipopolysaccharide LPS for 4 hours. At the same time, the inflammation was induced by intraperitoneal injection of LPS (LGG, ZJ617 or ZJ615) by intraperitoneal injection of 108 CFU/mL Lactobacillus (LGG, ZJ617 or ZJ615) for a week after construction of LPS inflammation model in the mice. The qRT-PCR technique was used to detect inflammatory cytokines and Toll like receptors (TLR) in IPEC-J2 cells and mice intestinal tissues. Level. Western-blot technique, immunohistochemistry and immunofluorescence technique were used to detect the expression level of major target proteins in IPEC-J2 cells and mouse intestinal tissues (NF- kappa B signaling pathway and MAPK signaling pathway). The results showed that: 1) LGG pretreated IPEC- compared with pure LPS stimulation of pig intestinal epithelial IPEC-J2 cells. J2 cells significantly reduced the LPS induced inflammatory cell factor IL-6, IL-12, TNF- alpha and TLR receptor TLR2, TLR4, TLR9 mRNA level, cell signaling molecule p38MAPK, ERK1/2 and nuclear kappa phosphorylation level significantly decreased. P and extracellular polysaccharide EPS pretreated IPEC-J2 cells can significantly reduce the LPS induced inflammatory cell factor IL-6, IL-12, TNF- alpha and TLR receptor TLR2, TLR4, TLR9 mRNA levels; Compared with the cells, the CpG ODN pretreated by LGG without methylated IPEC-J2 cells could significantly increase the LPS induced inflammatory cell factor IL-6, IL-12, TNF- alpha and TLR receptor TLR2, TLR4, TLR9. S stimulated the mice with LGG and low adhesion Lactobacillus ZJ615 for one week after the stimulation of LPS stimulation, and the inflammatory cytokine IL-6, IL-12, TNF- A and TLR receptor TLR2, TLR4, TLR9 mRNA levels in the intestinal tissue decreased significantly. The expression of I- kappa B alpha significantly increased (P0.05).5). Compared with the simple LPS stimulation of the mice without Lactobacillus irrigated the mice, the mice with high adhesion Lactobacillus ZJ617 received LPS stimulation for one week, and the inflammatory cytokines IL-6, IL-12, TNF- A and TLR receptor TLR2 in the intestinal tissue were significantly increased. At the same time, the mRNA level of anti inflammatory cytokine IL-10 in the intestinal tissue of mice was significantly increased, and the expression of I- kappa B alpha in the intestinal tissue was significantly decreased (P0.05). Conclusion: this can be seen from this conclusion: this can be seen that the Lactobacillus rhamnose LGG mainly inhibits MAPK and NF- kappa B signaling pathway by regulating the Toll like mRNA level in small intestinal epithelial cells. Live, reduce the level of inflammatory cytokine mRNA induced by LPS, and mediate intestinal immunoregulation. The surface protein SLP of Lactobacillus rhamnolipid LGG, extracellular polysaccharide EPS and CpG ODN have immune regulation effect on LPS induced intestinal epithelial cells, in which the non methylation CpG ODN has the immune stimulation effect. Different adhesion milk The intestinal mucosal immunoregulatory effects of acid bacilli were different. Among them, the low adhesion force Lactobacillus ZJ615 mediated intestinal immune regulation was similar to that of LGG.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816

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