發(fā)布時(shí)間：2018-06-05 23:53

  本文選題：炎癥性腸病 + 落新婦苷　； 參考：《揚(yáng)州大學(xué)》2014年碩士論文

【摘要】：[目的] 目前炎癥性腸病(IBD)的發(fā)病率呈逐年上升趨勢(shì),但其治療受限于有效率低、副作用大、價(jià)格昂貴等。落新婦苷,土茯苓中的主要單體,近來(lái)被報(bào)道是一種新型的免疫抑制劑。本研究主要明確落新婦苷是否具有抗右旋葡聚糖硫酸鈉(DSS)誘導(dǎo)的小鼠腸炎活性并探討其機(jī)制。 [方法] 本文采用DSS誘導(dǎo)的小鼠腸炎模型,予腹腔注射落新婦苷,觀察小鼠體重、血便、腹瀉等疾病活動(dòng)情況及腸道局部炎癥情況；采用酶聯(lián)免疫吸附實(shí)驗(yàn)檢測(cè)腸炎小鼠外周血中細(xì)胞因子的含量；流式細(xì)胞術(shù)檢測(cè)脾臟和腸道中樹(shù)突狀細(xì)胞(DCs)的表型、功能及調(diào)節(jié)性T細(xì)胞(Tregs)頻率的變化。不同濃度的落新婦苷刺激小鼠脾臟細(xì)胞,流式細(xì)胞術(shù)檢測(cè)其中DCs表型及功能的變化,并檢測(cè)Tregs頻率的變化。不同濃度的落新婦苷刺激RAW264.7細(xì)胞株,流式細(xì)胞術(shù)檢測(cè)胞內(nèi)活性氧(ROS)的水平和細(xì)胞因子(IL-1β, IL-12p40, IL-10, TGF-β, IL-15Rα, TNF-α)的分泌情況。取健康人及IBD患者外周血單核細(xì)胞(PBMC),經(jīng)細(xì)胞因子刺激誘導(dǎo)為未成熟的樹(shù)突狀細(xì)胞(imDCs),對(duì)比正常人及IBD患者DCs表型的差異,并檢測(cè)落新婦苷對(duì)IBD患者PBMC誘導(dǎo)的imDCs的表型和功能的變化。將落新婦苷預(yù)處理的小鼠髓樣DCs與CD4+T共孵育,檢測(cè)Tregs的頻率及CD4＋T細(xì)胞分泌IFN-γ的情況。 [結(jié)果] 1.落新婦苷可以抑制DSS腸炎小鼠的發(fā)病(體重、血便、腹瀉、腸道HE染色)； 2.落新婦苷作用后的腸炎小鼠外周血中IL-10、TGF-β的含量高于對(duì)照組； 3.落新婦苷體內(nèi)作用于腸炎小鼠,可促進(jìn)脾臟DCs分泌IL-10和TGF-β,抑制IL-12p40、IL-1p的分泌,下調(diào)脾臟DCs表面CD86的表達(dá),上調(diào)脾細(xì)胞中CD4+CD25+Foxp3+T細(xì)胞的頻率,對(duì)腸道DCs的表型無(wú)明顯影響。 4.不同濃度的落新婦苷刺激脾細(xì)胞可劑量依賴性的下調(diào)脾臟DCs表面CD86的表達(dá),對(duì)B7H1無(wú)明顯影響,促進(jìn)脾臟DCs分泌IL-10、TGF-β,抑制IL-1β、IL-12p40的分泌,上調(diào)脾細(xì)胞中CD4+CD25+Foxp3+T細(xì)胞的頻率； 5.落新婦苷抑制RAW264.7細(xì)胞胞內(nèi)活性氧的釋放,促進(jìn)IL-10、TGF-p的分泌,抑制IL-1β、IL-12p40的分泌,并且呈劑量依賴性； 6.IBD患者PBMC誘導(dǎo)imDCs的CD86的表達(dá)高于正常人,并且落新婦苷可以下調(diào)IBD患者PBMC誘導(dǎo)imDC的CD86的表達(dá),同時(shí)促進(jìn)imDCs分泌IL-10、TGF-p,抑制IL-1β、IL12p40的分泌； 7.經(jīng)落新婦苷預(yù)處理的小鼠髓樣DCs可以誘導(dǎo)CD4+CD25+Foxp3+T細(xì)胞的生成,抑制CD4＋T細(xì)胞分泌IFN-γ,并且該效應(yīng)可以被TGF-β1阻斷劑所逆轉(zhuǎn)。 [結(jié)論] 落新婦苷通過(guò)誘導(dǎo)DCs的免疫負(fù)向調(diào)節(jié)功能而發(fā)揮抗DSS腸炎小鼠的功效,同時(shí)落新婦苷可調(diào)節(jié)IBD患者PBMC來(lái)源DCs的免疫調(diào)節(jié)活性,經(jīng)落新婦苷刺預(yù)處理的DCs可以誘導(dǎo)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 偽, TNF- 偽 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- 緯 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- 緯 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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R574

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本文編號(hào)：1983982