【摘要】：乳酸桿菌是應(yīng)用最為廣泛的益生菌,在腸道穩(wěn)態(tài)調(diào)控中發(fā)揮重要作用。仔豬腸道應(yīng)激多發(fā),乳酸菌等益生菌對(duì)腸道的穩(wěn)恒調(diào)節(jié)起著重要的作用。因此,闡明乳酸桿菌對(duì)腸道免疫調(diào)節(jié)功能,解析其免疫調(diào)節(jié)的關(guān)鍵信號(hào)分子及信號(hào)網(wǎng)絡(luò),將為益生菌調(diào)控腸道穩(wěn)恒提供提供科學(xué)依據(jù),對(duì)仔豬腸道健康具有重要的意義。本試驗(yàn)以公認(rèn)的標(biāo)準(zhǔn)益生菌——鼠李糖乳酸桿菌(Lactobacillus rhamnousus GG,LGG)為代表,設(shè)計(jì)了兩個(gè)試驗(yàn)。試驗(yàn)一:構(gòu)建斷奶仔豬腸道炎癥模型,連續(xù)灌胃1x1010CFU/mL乳酸桿菌兩周,腹腔注射來(lái)源于大腸桿菌的脂多糖(LPS)來(lái)誘導(dǎo)炎癥,分析鼠李糖乳酸桿菌預(yù)處理對(duì)LPS誘導(dǎo)腸道炎癥的免疫調(diào)節(jié)作用及機(jī)制。實(shí)驗(yàn)二:以LGG為標(biāo)準(zhǔn)菌株,構(gòu)建小鼠炎癥模型,連續(xù)灌胃108CFU/mL鼠李糖乳酸桿菌兩周后,腹腔注射LPS誘發(fā)炎癥。采用Western blot技術(shù)、免疫組化技術(shù)檢測(cè)腸道組織中炎癥的相關(guān)信號(hào)通路關(guān)鍵蛋白的表達(dá)水平,采用高通量測(cè)序分析腸道的微生物群系,同時(shí)用氣相色譜檢測(cè)乳酸桿菌對(duì)代謝途徑的調(diào)節(jié)作用。實(shí)驗(yàn)一的研究結(jié)果表明::1)與單純LPS刺激仔豬相比,LGG預(yù)處理仔豬能顯著降低LPS誘導(dǎo)的細(xì)胞炎癥因子IL-6,TNF-α和顯著升高抗炎因子IL-10的表達(dá)量;細(xì)胞信號(hào)通路分子ERK1/2,p38MAPK和NF-κBp65的磷酸化水平顯著降低,但I(xiàn)-κBα表達(dá)量顯著升高(P0.05)。2)與單純LPS刺激仔豬相比,在屬水平上,LGG能夠減少梭菌、鏈球菌和乳桿菌,在門水平上能減少厚壁菌門和增加變形菌門。3)與對(duì)照相比,LPS能降低具有抗炎作用的熊果苷。實(shí)驗(yàn)二的結(jié)果表明:1)與單純LPS刺激小鼠相比,LGG連續(xù)灌胃2周的小鼠接受LPS刺激后,NF-κB基因敲除小鼠腸道炎癥細(xì)胞因子IL-6和TNF-α的表達(dá)量顯著升高;有趣的是p38基因敲除小鼠則出現(xiàn)相反的結(jié)果。2)與單純LPS刺激小鼠相比,野生型小鼠的細(xì)胞信號(hào)通路分子p38MAPK和NF-κBp65的磷酸化水平升高,與之前的研究結(jié)果相反,有趣的時(shí)NF-κB基因敲除小鼠的ERK1/2,p38MAPK和NF-κBp65的磷酸化水平顯著升高(P0.05)。p38基因敲除小鼠的ERK1/2,p38MAPK和NF-κBp65的磷酸化水平?jīng)]有顯著差異。6)與單純LPS刺激小鼠相比,在屬水平上,LGG能夠減少賀氏菌屬,在門水平上能減少變形菌門和增加變形菌門厚壁菌門。由此可見,鼠李糖乳酸桿菌LGG能夠?qū)PS誘導(dǎo)的腸道炎癥發(fā)揮免疫調(diào)節(jié)作用,通過(guò)抑制細(xì)胞MAPK和NF-κB信號(hào)通路激活,降低炎癥因子,從而介導(dǎo)腸道免疫調(diào)節(jié)作用。
[Abstract]:Lactobacillus is the most widely used probiotics and plays an important role in the regulation of intestinal homeostasis. The intestinal stress of piglets is frequent, and probiotics such as lactic acid bacteria play an important role in the stable regulation of intestinal tract. Therefore, to elucidate the function of Lactobacillus on intestinal immune regulation and to analyze its key signal molecules and signal networks will provide a scientific basis for probiotic regulation of intestinal stability and play an important role in the intestinal health of piglets. In this experiment, two experiments were designed, represented by the standard probiotics-Lactobacillus rhamnoides (Lactobacillus rhamnousus GG,LGG). Experiment 1: the model of intestinal inflammation in weaned piglets was established, and the inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli for two weeks by intragastric administration of Lactobacillus 1x1010CFU/mL. To analyze the immunomodulatory effect and mechanism of lactobacillus rhamnoides pretreatment on intestinal inflammation induced by LPS. Experiment 2: using LGG as the standard strain, the inflammatory model of mice was established. After two weeks of continuous intragastric administration of Lactobacillus rhamnoides 108CFU/mL, LPS was injected intraperitoneally to induce inflammation. Western blot technique and immunohistochemical technique were used to detect the expression of key proteins related to inflammatory signaling pathway in intestinal tissue. High throughput sequencing was used to analyze the microflora of intestinal tract. At the same time, the regulation of Lactobacillus on metabolic pathway was detected by gas chromatography. The results of experiment 1 showed that: 1) compared with LPS alone, LGG pretreated piglets could significantly reduce the expression of LPS induced cytokine IL-6,TNF- 偽 and increase the expression of anti-inflammatory factor IL-10; The phosphorylation levels of ERK1/2,p38MAPK and NF- 魏 Bp65 decreased significantly, but the expression of I- 魏 B 偽 increased significantly (P0.05). Compared with LPS alone, LGG decreased the levels of clostridium, streptococcus and lactobacillus. Compared with the control, LPS could reduce the arbutin with anti-inflammatory effect. The results of experiment 2 showed that: 1) the expression of NF- 魏 B gene knockout inflammatory cytokines IL-6 and TNF- 偽 in NF- 魏 B knockout mice was significantly higher than that in mice stimulated by LPS for 2 weeks. Interestingly, p38 knockout mice showed the opposite results. 2) the phosphorylation levels of p38MAPK and NF- 魏 Bp65 in wild-type mice were higher than those in mice stimulated by LPS alone, contrary to previous studies. Interestingly, the phosphorylation levels of ERK1/2,p38MAPK and NF- 魏 Bp65 in NF- 魏 B knockout mice were significantly increased (P0.05). There was no significant difference in phosphorylation levels between ERK1/2,p38MAPK and NF- 魏 Bp65 in p38 knockout mice. At the gate level, it can reduce the phylum of Proteus and increase the phylum of the phylum. It can be concluded that Lactobacillus rhamnoides LGG can play an immunomodulatory role on the intestinal inflammation induced by LPS. By inhibiting the activation of MAPK and NF- 魏 B signaling pathway, and reducing the inflammatory factors, Lactobacillus rhamnoides can mediate the intestinal immune regulation.
【學(xué)位授予單位】：浙江農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S816

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