双歧杆菌及其完整肽聚糖对脐血来源树突状细胞免疫调节功能影响的实验研究

发布时间：2018-05-25 15:26

本文选题：双歧杆菌完整肽聚糖 + 树突状细胞　；参考：《重庆医科大学》2008年硕士论文

【摘要】： 目的:通过研究双歧杆菌和不同剂量双歧杆菌完整肽聚糖(Whole Peptidoglycan, WPG)对脐血单核细胞来源树突状细胞(Cord blood monocyte-derived dendritic cells, CB-MDDCs)形态、表面标志、刺激淋巴细胞增殖功能及分泌细胞因子的影响,了解双歧杆菌及其WPG在体外对CB-MDDCs分化、成熟及免疫调节功能的作用;探讨双歧杆菌防治免疫失调性疾病的可能机制,为益生菌及其生物活性成分的进一步开发提供依据。方法:分离正常孕妇脐血单个核细胞(Cord blood monocular cell,CBMC),贴壁培养获得脐血单核细胞(Cord blood Monocyte, CB-Mon), GM-CSF、IL-4联合诱导单核细胞生成未成熟树突状细胞(Dendritic cells, DCs),实验组在培养的第7天分别加入两歧双歧杆菌全菌、不同剂量(1μg/ml-8μg/ml)两歧双歧杆菌WPG,阳性对照组加入脂多糖(Lipopolysaccharide,LPS),阴性对照组仅加入培养基。通过CB-MDDCs形态学观察,流式细胞术检测CB-MDDCs表面标志物CD83及CD1a的表达,MLR检测CB-MDDCs刺激同种异体T淋巴细胞能力,ELISA法测定CB-MDDCs培养上清中IL-12、IL-10的分泌,确定双歧杆菌及其WPG在体外对CB-MDDCs成熟程度及功能的影响;比较双歧杆菌与其WPG的免疫调节作用的差别。结果:(1)CB-Mon在双歧杆菌或其WPG(1-6μg/ml)与GM-CSF、IL-4协同诱导作用下,成为形态上具有典型树突状突起的DCs;诱导后的CB-MDDCs刺激同种异体T细胞的增殖能力及分泌IL-12p70、IL-10的水平显著高于阴性对照组(P0.01);其中以WPG 5μg/ml组刺激能力最强,且该组细胞表面标志物CD83及CD1a的表达增加。(2)当WPG的量达到2μg/ml时,CB-MDDCs刺激同种异体T淋巴细胞的能力及分泌IL-12p70、IL-10的水平与双歧杆菌全菌组没有统计学的差异(P0.05),而当WPG的量达到5μg/ml时,其对CB-MDDCs的刺激能力则明显高于双歧杆菌全菌组(P0.01);(3)WPG的浓度在1- 5μg/ml时CB-MDDCs分泌的IL-12及IL-10的量呈剂量依赖性,其中WPG 5μg/ml时作用最为显著,WPG量为6μg/ml时分泌的量减少,WPG量为8μg/ml时,分泌的量与阴性对照组无差异(P0.05)。结论: 1.双歧杆菌及其WPG能影响CB-MDDCs的成熟状态。 2.双歧杆菌及其WPG通过刺激CB-MDDCs分泌细胞因子调节Th1/Th2之间平衡,可能是防治免疫失调性疾病的作用机制之一。 3.双歧杆菌WPG对CB-MDDCs成熟程度及分泌细胞因子水平的影响强于双歧杆菌全菌,说明WPG是双歧杆菌主要免疫活性成分;不同剂量WPG的免疫调节作用呈一定量效关系,提示益生菌制剂只能在一定剂量范围内发挥有益作用。
[Abstract]:Objective: to study the effects of Bifidobacterium and different doses of Bifidobacterium peptidoglycan (WPG) on the morphology, surface markers, lymphocyte proliferation and cytokine secretion of cord blood monocyte derived dendritic cells (CB-MDDCs). To understand the effects of bifidobacterium and its WPG on the differentiation, maturation and immunomodulation of CB-MDDCs in vitro, and to explore the possible mechanism of bifidobacterium in preventing and treating immune disorders, which provides the basis for the further development of probiotics and its bioactive components. Methods: umbilical cord blood mononuclear cells (Cord blood monocular cells) were isolated from normal pregnant women, and cord blood monocytes, CB-Monocyte, CB-Monocyte, GM-CSFFN- IL-4 were isolated from cord blood mononuclear cells to induce the formation of immature dendritic cells (DCS). The experimental group was cultured on the 7th day. Add Bifidobacterium bifidobacterium, Different doses of 1 渭 g/ml-8 渭 g / ml of bifidobacterium (WPGs) were added to the lipopolysaccharide of lipopolysaccharide in the positive control group, and only to the medium in the negative control group. The expression of CD83 and CD1a on the surface of CB-MDDCs was detected by flow cytometry. The ability of CB-MDDCs to stimulate allogeneic T lymphocytes was detected by Elisa. The secretion of IL-12 and IL-10 in the supernatant of CB-MDDCs was determined by Elisa. To determine the effect of Bifidobacterium and its WPG on the maturation and function of CB-MDDCs in vitro, and to compare the immunomodulatory effect between Bifidobacterium and its WPG. Results the CB-Mon was induced by Bifidobacterium or its WPG(1-6 渭 g / ml in collaboration with GM-CSFU IL-4. The ability of CB-MDDCs to stimulate the proliferation of allogeneic T cells and the level of IL-12p70 ~ + IL-10 secretion were significantly higher than those of negative control group (P 0.01), and the stimulation ability of WPG 5 渭 g/ml group was the highest. Moreover, the expression of CD83 and CD1a on the cell surface was increased.) when the amount of WPG reached 2 渭 g/ml, the ability of CB-MDDCs to stimulate allogeneic T lymphocytes and the level of IL-12p70 IL-10 secreted by CB-MDDCs were not significantly different from those of the whole bifidobacterium group, but when the amount of WPG reached 5 渭 g/ml, there was no significant difference between them. Its stimulating ability to CB-MDDCs was significantly higher than that of P0.01M3WPG of Bifidobacterium in a dose-dependent manner when the concentration of CB-MDDCs was 1 -5 渭 g/ml, and the amount of IL-12 and IL-10 secreted by CB-MDDCs was most significant when WPG 5 渭 g/ml was 6 渭 g/ml, and the quantity of WPG decreased to 8 渭 g/ml when the concentration of WPG 5 渭 g/ml was 6 渭 g/ml. There was no difference in secretion between the control group and the negative control group (P 0.05). Conclusion: 1. Bifidobacterium and its WPG can affect the mature state of CB-MDDCs. 2. Bifidobacterium and its WPG regulate the balance of Th1/Th2 by stimulating the secretion of cytokines by CB-MDDCs, which may be one of the mechanisms of preventing and treating immune disorders. 3. The effect of Bifidobacterium WPG on CB-MDDCs maturation and cytokine secretion was stronger than that of Bifidobacterium, indicating that WPG was the main immune active component of Bifidobacterium, and the immunomodulatory effect of different doses of WPG was in a dose-dependent manner. The results suggest that probiotics can only play a beneficial role in a certain dose range.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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