落新妇苷对树突状细胞的免疫调节活性及其抗炎症性肠病的机制研究

发布时间：2018-06-05 23:53

本文选题：炎症性肠病 + 落新妇苷　；参考：《扬州大学》2014年硕士论文

【摘要】：[目的] 目前炎症性肠病(IBD)的发病率呈逐年上升趋势,但其治疗受限于有效率低、副作用大、价格昂贵等。落新妇苷,土茯苓中的主要单体,近来被报道是一种新型的免疫抑制剂。本研究主要明确落新妇苷是否具有抗右旋葡聚糖硫酸钠(DSS)诱导的小鼠肠炎活性并探讨其机制。 [方法] 本文采用DSS诱导的小鼠肠炎模型,予腹腔注射落新妇苷,观察小鼠体重、血便、腹泻等疾病活动情况及肠道局部炎症情况；采用酶联免疫吸附实验检测肠炎小鼠外周血中细胞因子的含量；流式细胞术检测脾脏和肠道中树突状细胞(DCs)的表型、功能及调节性T细胞(Tregs)频率的变化。不同浓度的落新妇苷刺激小鼠脾脏细胞,流式细胞术检测其中DCs表型及功能的变化,并检测Tregs频率的变化。不同浓度的落新妇苷刺激RAW264.7细胞株,流式细胞术检测胞内活性氧(ROS)的水平和细胞因子(IL-1β, IL-12p40, IL-10, TGF-β, IL-15Rα, TNF-α)的分泌情况。取健康人及IBD患者外周血单核细胞(PBMC),经细胞因子刺激诱导为未成熟的树突状细胞(imDCs),对比正常人及IBD患者DCs表型的差异,并检测落新妇苷对IBD患者PBMC诱导的imDCs的表型和功能的变化。将落新妇苷预处理的小鼠髓样DCs与CD4+T共孵育,检测Tregs的频率及CD4＋T细胞分泌IFN-γ的情况。 [结果] 1.落新妇苷可以抑制DSS肠炎小鼠的发病(体重、血便、腹泻、肠道HE染色)； 2.落新妇苷作用后的肠炎小鼠外周血中IL-10、TGF-β的含量高于对照组； 3.落新妇苷体内作用于肠炎小鼠,可促进脾脏DCs分泌IL-10和TGF-β,抑制IL-12p40、IL-1p的分泌,下调脾脏DCs表面CD86的表达,上调脾细胞中CD4+CD25+Foxp3+T细胞的频率,对肠道DCs的表型无明显影响。 4.不同浓度的落新妇苷刺激脾细胞可剂量依赖性的下调脾脏DCs表面CD86的表达,对B7H1无明显影响,促进脾脏DCs分泌IL-10、TGF-β,抑制IL-1β、IL-12p40的分泌,上调脾细胞中CD4+CD25+Foxp3+T细胞的频率； 5.落新妇苷抑制RAW264.7细胞胞内活性氧的释放,促进IL-10、TGF-p的分泌,抑制IL-1β、IL-12p40的分泌,并且呈剂量依赖性； 6.IBD患者PBMC诱导imDCs的CD86的表达高于正常人,并且落新妇苷可以下调IBD患者PBMC诱导imDC的CD86的表达,同时促进imDCs分泌IL-10、TGF-p,抑制IL-1β、IL12p40的分泌； 7.经落新妇苷预处理的小鼠髓样DCs可以诱导CD4+CD25+Foxp3+T细胞的生成,抑制CD4＋T细胞分泌IFN-γ,并且该效应可以被TGF-β1阻断剂所逆转。 [结论] 落新妇苷通过诱导DCs的免疫负向调节功能而发挥抗DSS肠炎小鼠的功效,同时落新妇苷可调节IBD患者PBMC来源DCs的免疫调节活性,经落新妇苷刺预处理的DCs可以诱导Tregs的生成。
[Abstract]:[purpose] At present, the incidence of inflammatory bowel disease (IBD) is increasing year by year, but its treatment is limited by low effective rate, big side effect, high price and so on. Acanthoside, the main monomer in Poria cocos, has recently been reported as a novel immunosuppressant. The purpose of this study was to investigate whether Acanthoside has the activity of anti-DSS-induced enteritis in mice and to explore its mechanism. [methods] The mouse enteritis model induced by DSS was injected intraperitoneally to observe the activity of body weight, blood stool, diarrhea and local inflammation of intestine. The levels of cytokines in peripheral blood of mice with enteritis were detected by enzyme linked immunosorbent assay (Elisa), and the phenotype, function and Tregs frequency of dendritic cells in spleen and intestine were detected by flow cytometry. The spleen cells of mice were stimulated with different concentrations of asparagine. The phenotype and function of DCs and the frequency of Tregs were detected by flow cytometry. The levels of intracellular reactive oxygen species (Ros) and the secretion of cytokines such as IL-1 尾, IL-12p40, IL-10, TGF- 尾, IL-15R 伪, TNF- 伪 were detected by flow cytometry. Peripheral blood mononuclear cells (PBMC) from healthy people and patients with IBD were induced into immature dendritic cells by cytokine stimulation. The phenotypic differences of DCs were compared between normal subjects and IBD patients. The phenotypic and functional changes of imDCs induced by PBMC in patients with IBD were detected. Mouse myeloid DCs pretreated with Acanthrin was co-incubated with CD4 T to detect the frequency of Tregs and the secretion of IFN- 纬 by CD4+T cells. [results] 1. Asparagin could inhibit the development of DSS enteritis mice (body weight, blood stool, diarrhea, intestinal HE staining). 2. The level of IL-10 TGF- 尾 in peripheral blood of mice with enteritis treated by Acanthrin was higher than that of control group. 3. In mice with enteritis, the effect of Agnin on the secretion of IL-10 and TGF- 尾 by DCs in spleen inhibited the secretion of IL-12p40 and IL-1p, down-regulated the expression of CD86 on the surface of DCs, upregulated the frequency of CD4 CD25 Foxp3 T cells in splenocytes, and had no significant effect on the phenotype of intestinal DCs. 4. Different concentrations of Agnin stimulated splenocytes could down-regulate the expression of CD86 on spleen DCs surface in a dose-dependent manner, but had no effect on B7H1. It could promote the secretion of IL-10TGF- 尾 by DCs, inhibit the secretion of IL-12p40 by IL-1 尾, and upregulate the frequency of CD4 CD25 Foxp3 T cells in splenocytes. 5. Acanthrin inhibited the release of reactive oxygen species in RAW264.7 cells, promoted the secretion of IL-10 TGF-p, and inhibited the secretion of IL-12p40 of IL-1 尾 in a dose-dependent manner. The expression of CD86 in imDCs induced by PBMC in 6.IBD patients was higher than that in normal controls, and the expression of imDC CD86 induced by PBMC in IBD patients was down-regulated, and imDCs secreted IL-10 TGF-pand inhibited the secretion of IL-1 尾 -IL-12p40. 7. Mouse myeloid DCs pretreated with asparagine could induce the production of CD4 CD25 Foxp3 T cells and inhibit the secretion of IFN- 纬 by CD4+T cells, which could be reversed by TGF- 尾 1 blocker. [conclusion] Acanthrin can inhibit DSS enteritis mice by inducing the negative regulation function of DCs, and can regulate the immunomodulatory activity of DCs derived from PBMC in IBD patients. DCs pretreated with IBD can induce the production of Tregs.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R574

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