本文選題：乳酸桿菌 + LGG��； 參考：《浙江農(nóng)林大學》2015年碩士論文

【摘要】：益生菌廣泛用于調(diào)節(jié)動物腸道健康,乳酸桿菌為應用最早、研究最多的益生菌菌種之一。乳酸桿菌通過其不同成分能夠介導動物胃腸道免疫調(diào)節(jié)作用,發(fā)揮益生效應,然而其免疫調(diào)節(jié)機制仍不明了。鑒于此,闡明乳酸桿菌具有怎么樣的腸道免疫調(diào)節(jié)功能,解析其免疫調(diào)節(jié)的信號通路及其關(guān)鍵靶點,將為益生菌免疫調(diào)節(jié)功能的開發(fā)提供科學依據(jù),對篩選和培育高效乳酸桿菌等益生菌具有重要的實踐意義。本試驗旨在以公認的標準益生乳酸桿菌——鼠李糖乳酸桿菌(Lactobacillus rhamnosus GG,LGG)為代表,同時分離并獲得LGG不同成分(表面蛋白SLP、胞外多糖EPS、基因組DNA以及人工合成的未甲基化CpG ODN),分析LGG及其不同成分對人工致敏體外培養(yǎng)腸上皮細胞的免疫調(diào)節(jié)效應和免疫保護作用,查明乳酸桿菌對小腸上皮細胞信號通路的調(diào)控作用,解析乳酸桿菌實現(xiàn)免疫調(diào)節(jié)作用的信號通路靶點,闡明乳酸桿菌發(fā)揮腸道免疫調(diào)控作用的分子機制。與此同時,以LGG作為參考菌株,構(gòu)建小鼠炎癥模型研究豬源乳酸桿菌(高粘附菌株Lactobacillus reuteri ZJ617和低粘附菌株Lactobacillus reuteri ZJ615)對腸道的免疫調(diào)節(jié)作用,分析乳酸桿菌介導動物腸道免疫調(diào)節(jié)作用的機制。本研究主要采用大腸桿菌LPS分別誘導豬小腸上皮細胞系IPEC-J2細胞和C57BL/6小鼠產(chǎn)生免疫反應,構(gòu)建炎癥模型以評定乳酸桿菌的免疫調(diào)節(jié)作用。通過構(gòu)建豬小腸上皮細胞系IPEC-J2細胞脂多糖LPS炎癥模型,采用2×107 CFU/mL LGG或從LGG中分離純化獲得的不同成分(表面蛋白SLP、基因組DNA、胞外多糖EPS以及人工合成的未甲基化CpG ODN序列),分別預處理IPEC-J2細胞(1×106細胞/孔)4小時,再以脂多糖LPS刺激細胞。同時,通過構(gòu)建C57BL/6小鼠LPS炎癥模型,連續(xù)灌胃108 CFU/mL乳酸桿菌(LGG、ZJ617或ZJ615)一周后,腹腔注射LPS誘發(fā)炎癥。采用qRT-PCR技術(shù)檢測IPEC-J2細胞和小鼠腸道組織致炎性細胞因子以及Toll樣受體(TLR)的mRNA水平。采用Western-blot技術(shù)、免疫組化和免疫熒光技術(shù)分別檢測IPEC-J2細胞和小鼠腸道組織中炎癥相關(guān)信號通路(NF-κB信號通路和MAPK信號通路)主要靶點蛋白的表達水平。研究結(jié)果表明:1)與單純LPS刺激豬腸上皮IPEC-J2細胞相比,LGG預處理IPEC-J2細胞后顯著降低由LPS誘導的細胞致炎性細胞因子IL-6、IL-12、TNF-α和TLR受體TLR2、TLR4、TLR9的mRNA水平;細胞信號通路分子p38MAPK、ERK1/2和NF-κBp65的磷酸化水平顯著降低,而I-κBα表達量顯著上升(P0.05)。2)與單純LPS刺激IPEC-J2細胞相比,LGG表面蛋白SLP和胞外多糖EPS預處理IPEC-J2細胞后能夠顯著降低由LPS誘導的細胞致炎性細胞因子IL-6、IL-12、TNF-α和TLR受體TLR2、TLR4、TLR9的mRNA水平;細胞信號通路分子p38MAPK和NF-κBp65磷酸化水平顯著降低,而I-κBα表達量顯著上升(P0.05)。3)與單純LPS刺激IPEC-J2細胞相比,LGG未甲基化的CpG ODN預處理IPEC-J2細胞能夠顯著升高由LPS誘導的細胞致炎性細胞因子IL-6、IL-12、TNF-α水平和TLR受體TLR2、TLR4、TLR9的mRNA水平;信號通路分子p38MAPK、ERK1/2磷酸化水平與單純LPS刺激細胞相比沒有顯著差異(P0.05)。4)與單純LPS刺激未經(jīng)乳酸桿菌灌胃小鼠相比,LGG和低粘附力乳酸桿菌ZJ615連續(xù)灌胃一周的小鼠接受LPS刺激后,其腸道組織中炎性細胞因子IL-6、IL-12、TNF-α和TLR受體TLR2、TLR4、TLR9的mRNA水平顯著下降;腸道組織中信號通路分子p38MAPK、ERK1/2和NF-κBp65的磷酸化水平顯著降低,而I-κBα表達量顯著上升(P0.05)。5)與單純LPS刺激未經(jīng)乳酸桿菌灌胃小鼠相比,高粘附力乳酸桿菌ZJ617連續(xù)灌胃一周的小鼠接受LPS刺激后,其腸道組織中炎性細胞因子IL-6、IL-12、TNF-α和TLR受體TLR2、TLR4、TLR9的mRNA水平顯著升高,同時小鼠腸道組織中抗炎性細胞因子IL-10的mRNA水平顯著升高;腸道組織中信號通路分子I-κBα表達量顯著下降(P0.05)。研究結(jié)論:由此可見,鼠李糖乳酸桿菌LGG主要通過調(diào)節(jié)小腸上皮細胞Toll樣受體mRNA水平,抑制細胞MAPK和NF-κB信號通路激活,降低LPS誘導的炎性細胞因子mRNA水平,從而介導腸道免疫調(diào)節(jié)作用。鼠李糖乳酸桿菌LGG成分表面蛋白SLP、胞外多糖EPS以及未甲基化CpG ODN對LPS誘導的腸上皮細胞具有免疫調(diào)控作用,其中未甲基化CpG ODN具有免疫刺激作用。不同粘附力乳酸桿菌腸道免疫調(diào)節(jié)效應存在差異,其中低粘附力乳酸桿菌ZJ615介導腸道免疫調(diào)控作用與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.
【學位授予單位】：浙江農(nóng)林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S816

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