【作者】 李丹；

【导师】 李艾黎；

【作者基本信息】 东北农业大学 ， 食品科学， 2014， 硕士

【摘要】 肽聚糖(whole peptidoglycan, WPG)是乳杆菌细胞壁中的重要组分,新的研究指出乳酸菌的免疫赋活功能与WPG调节Toll样受体2/对核因子(TLR2/NF-κB)信号通路密切相关,但对乳杆菌是否通过该机制缓解牛乳过敏尚不清楚。故本实验提取可显著抑制p-乳球蛋白(β-Lg)过敏的嗜酸乳杆菌的WPG成分,首先通过细胞实验,分析嗜酸乳杆菌WPG对过敏小鼠脾淋巴细胞抗体分泌、淋巴细胞CD4+/CD8+比值、Th1/Th2、Treg/Th17型细胞因子生成及其mRNA表达的影响;进而构建β-Lg过敏动物模型,分析嗜酸乳杆菌WPG调节TLR2/NF-κB信号通路对β-Lg过敏发生发展的影响,并探讨嗜酸乳杆菌WPG调节TLR2/NF-κB信号通路与Treg/Th17平衡的相关性,研究将揭示乳酸菌抑制过敏的作用途径及其信号识别的分子机制,为益生菌及其制剂的开发利用提供依据。一、细胞培养实验1.分析不同浓度嗜酸乳杆菌WPG对小鼠原代脾淋巴细胞增殖及转化能力的影响。结果显示当WPG浓度在10-1000mg/L范围时能显著促进小鼠体外脾淋巴细胞增殖及转化(P<0.05),且表现出剂量依赖关系。2.分析不同剂量嗜酸乳杆菌WPG对致敏小鼠脾淋巴细胞亚型、Th1/Th和Treg/Th17型细胞因子及抗体分泌的影响。结果表明,WPG体外刺激致敏脾细胞后可显著抑制IgE和β-Lg-特异性IgE的产生,上调CD3+、CD4+细胞数及CD4+/CD8+比值,平衡T淋巴细胞亚群数量,并通过提高T-bet/GATA-3及Foxp3/RORyt比例来促进Th1/Th2及Treg/Th17细胞平衡,且与过敏组相比,中、高剂量WPG的作用效果显著(P<0.05)。二、动物实验分析不同剂量嗜酸乳杆菌WPG对致敏小鼠Treg/Th17细胞失衡及TLR2/NF-κB信号通路的影响。结果表明嗜酸乳杆菌WPG干预可有效缓解p-Lg过敏,与过敏组小鼠比较,中剂量(20mg/kg wt)及高剂量(20mg/kg wt)组小鼠血清总IgE和(β-Lg-sIgE水平明显降低(P<0.05)；同时不同剂量WPG均能够显著上调Treg型细胞因子及转录因子(TGF-β、Foxp3、CD25)的表达,而中高剂量WPG对Th17型因子(IL-17、RORyt)及炎性因子(IL-6、IL-23)的表达的下调作用较为明显,进而纠正Treg/Th17细胞失衡。此外,与过敏组相比,中及高剂量WPG组中小鼠脾脏TLR2/NF-κB信号被明显激活,且具有剂量依赖性。特别是在高剂量组中,Foxp3的基因表达与TLR2/NF-κB信号表达成正相关,而RORγt与其信号通路成负关性,且相关性显著(P<0.05)。本实验表明嗜酸乳杆菌WPG成分对β-Lg过敏症状具有一定抑制作用,其免疫机制可能与WPG激活TLR2/NF-κB信号转导通路,进而平衡T淋巴细胞亚群数量,调节Th1/Th2及Treg/Th17细胞失衡密切相关。更多还原

【Abstract】 New data showed that whole peptidoglycan (WPG), as a critical component of cell wall from Lactobacillus acidopHilus(L.acidophilus), was possibly correlated with the immunomodulatory effects of probiotics via Toll-like receptor (TLR)2/NF-kappaB (NF-κB) signaling pathway. However, it is unclear whether the anti-allergy ability of L.acidophilus was attributed to TLR2/NF-KB pathway. Therefore, WPG from L.acidophilus with significantly inhibited function on β-Lg allergy was prepared as the research object. Screening platform for cell immunity was established at first, the effects of the different doses of WPG from L.acidophilus on the lymphocyte proliferation, antibody and cytokines production, CD4+/CD8+ratio, cytokine and gene expression of T-helper (Th)1/Th2and regulatory T cells (Treg)/Th17balance were observed; Animal models were further established using β-Lg as allergy. We administered allergic mice with varying doses of WPG to observe the effects of TLR2/NF-κB signaling pathway on the development of allergy, and investigate the correlations between TLR2/NF-κB and Treg/Th17imbalance. The results may reflect a new protective mechanism of the Lactobacilli to limit excessive inflammation and collateral tissue damage during allergy, and provid theoretical basises for screening and usage of anti-allergic Lactobacillus.The first experiment:Cell culture experiment.1. To observe the effects of different dose of WPG from Lactobacillus on the lymphocyte proliferation in vitro. WPG could both accelerate proliferation of splenocytes directly and show a dose-dependent relationship (P<0.05).2. Effect of different Lactobacillus on splenic subtype, Th1/Th2and Treg/Th17balance, and antibody production of allergic splenic lymphocyte in mice in vitro. Compared with the allergy group, the secretion of total IgE and β-Lg-specific IgE were decreased in the splenocytes stimulated with WPG in a dose manner; In addition, the numbers of CD3+and CD4+cells were significantly promoted by WPG, as well as and the ratio of CD4+/CD+cells in order to maintain the balance of T lymphocyte subsets; WPG administration significantly enhanced the gene ratio of T-bet/GATA-3and Foxp3/RORyt to regulate Th1/Th2and Treg/Th17balance, especially in medium and high dose group.The second experiment:Animal experiment.To analyze the effects of different dose of WPG from Lactobacillus on the imbalance of Treg/Th17and TLR2/NF-κB signaling pathway.The results indicated that intraperitoneal injection of medium and high dose of WPG had an ability to effectively inhibit IgE-induced β-Lg allergy compared with allergy group (P<0.05); Meanwhile, treatment with different doses of WPG increased the levels of Treg-type cytokine and transcription factor (TGF-P, Foxp3, CD25), but decreased those of Th17(IL-17, RORyt) and inflammatory cytokines (IL-6, IL-23) in medium and high dose group to improved the Th17/Treg balance. Furthermore, compared with allergy group, TLR2/NF-κB signialling were clearly activated in spleen of WPG group in a dose manner, especially positive correlated with the Foxp3expression, but negative correlated with the RORyt expression in WPG-treated group (P<0.05).On this study, WPG form L.acidophilus have the effects on release β-Lg allergy, which may attribute to TLR2/NF-κB signaling pathway, leading to the balance of splenic subtypes, Thl/Th2and Treg/Th17cell balance.更多还原