【摘要】：研究背景炎癥性腸病(Inflammatory bowel disease,IBD),是一類主要影響人類胃腸道的慢性疾病,包括潰瘍性結(jié)腸炎和克羅恩病。雖然其具體的病理機制尚未完全明確,但現(xiàn)有的證據(jù)表明腸粘膜免疫調(diào)控紊亂和上皮屏障功能障礙共同引發(fā)了炎癥性腸病的發(fā)生。芳香烴受體(AhR)是一種胞漿內(nèi)的轉(zhuǎn)錄因子,可由一系列的外界環(huán)境中的配體特異性激活。AhR的激活參與腸粘膜免疫的調(diào)控。有證據(jù)表明,在IBD病人的腸道組織中AhR的表達被抑制,而給予AhR的配體6-甲�；胚岵3,2b]-咔唑(FICZ)和四氯二苯并-p-二惡英(TCDD)可以緩解小鼠結(jié)腸炎模型中的炎癥反應。雖然現(xiàn)有的一些研究已經(jīng)證實了AhR在炎癥性腸病中的保護作用,但是其作用的具體機制尚未明確。白介素10(IL-10)是一種重要的抗炎因子,可以緩解腸粘膜炎癥。本研究通過外源性給予AhR的特異性激動劑FICZ,研究活化的AhR對DSS誘導的小鼠結(jié)腸炎的保護作用并探討其機制是否是通過上調(diào)腸上皮源IL-10的表達來實現(xiàn)的。研究方法1.給予小鼠4%的DSS喂養(yǎng)7天誘導小鼠結(jié)腸炎模型,兩天后腹腔注射AhR激動劑FICZ。每天記錄小鼠體重,第8天收集小鼠結(jié)腸組織并測量長度,切片并進行HE染色和組織學分析。2.從結(jié)腸組織中提取腸上皮細胞,用免疫印跡技術檢測腸上皮細胞中IL-10的表達,用實時熒光定量PCR技術檢測炎性因子mRNA的表達。3.在體外培養(yǎng)Caco-2細胞株,用FICZ處理細胞,或同時給予脂多糖(LPS)處理,用免疫印跡技術檢測IL-10和STAT3/P-STAT3的表達,用用實時熒光定量PCR技術檢測炎性因子mRNA的表達。研究結(jié)果1.給予FICZ明顯緩解了DSS引起的小鼠體重下降;DSS處理后小鼠結(jié)腸的長度幾乎縮短一半并伴有水腫和淤血,而給予FICZ后這些情況明顯改善;組織學分析顯示DSS處理后小鼠結(jié)腸上皮和隱窩有嚴重的缺損,而FICZ處理后明顯改善了這些損傷。2.與DSS處理組相比,給予FICZ明顯上調(diào)了腸上皮源IL-10的表達;DSS處理后,促炎的炎性因子的mRNA表達明顯增高,而給予FICZ處理后炎性因子明顯下調(diào)。3.體外培養(yǎng)Caco2細胞,給予LPS處理24小時或LPS和FICZ共同處理24小時,LPS明顯上調(diào)了IL-1β和IL-6的mRNA的表達,而FICZ明顯下調(diào)其表達;同時無論是否有LPS的刺激,FICZ均明顯促進了IL-10在蛋白水平和mRNA水平的表達。4.FICZ處理Caco-2細胞24小時后,STAT3的表達幾乎沒有變化而P-STAT3的表達明顯上調(diào)。結(jié)論1.FICZ激活AhR明顯緩解DSS誘導的小鼠結(jié)腸炎。2.FICZ激活AhR在動物實驗和細胞實驗均可以降低炎性因子的表達。3.活化的AhR可以上調(diào)腸上皮細胞源IL-10和P-STAT3的表達。
[Abstract]:Background: inflammatory bowel disease (Inflammatory bowel) is a class of chronic diseases mainly affecting human gastrointestinal tract, including ulcerative colitis and Crohn's disease. Although its specific pathological mechanism has not been completely clear, the existing evidence shows that intestinal mucosal immune regulation disorder and epithelial barrier dysfunction jointly lead to the occurrence of inflammatory bowel disease. Aromatics receptor (AhR) is a transcriptional factor in the cytoplasm, which can be activated by a series of ligands in external environment. AhR activation is involved in the regulation of intestinal mucosal immunity. It has been shown that the expression of AhR was inhibited in the intestinal tissues of IBD patients, while the ligands 6-formylindoledo [3b2b] -carbazole (FICZ) and tetrachlorodibenzo-pdioxin (TCDD), which were administered with AhR, could attenuate the inflammatory reaction in mice colitis model. Although some existing studies have confirmed the protective role of AhR in inflammatory bowel disease, the specific mechanism of its role has not been clarified. Interleukin 10 (IL-10) is an important anti-inflammatory factor, which can relieve intestinal mucosal inflammation. In this study, the protective effect of activated AhR on DSS induced colitis in mice was studied by exogenous administration of FICZ, a specific agonist of AhR, and the mechanism was investigated by up-regulating the expression of IL-10 in intestinal epithelium. Method 1. Mice were fed with 4% DSS for 7 days to induce colitis. Two days later, AhR agonist FICZ was injected intraperitoneally. The body weight of mice was recorded daily. The colonic tissues were collected and measured on the 8th day, sections were made and HE staining and histological analysis were performed. Intestinal epithelial cells were extracted from colon tissue. The expression of IL-10 in intestinal epithelial cells was detected by Western blotting, and the expression of inflammatory factor mRNA was detected by real-time fluorescence quantitative PCR. Caco-2 cell lines were cultured in vitro. The cells were treated with FICZ or treated with lipopolysaccharide (LPS). The expression of IL-10 and STAT3/P-STAT3 was detected by Western blot and the expression of inflammatory factor mRNA was detected by real-time fluorescence quantitative PCR technique. Results 1. The colonic length of mice treated with FICZ significantly alleviated the weight loss induced by DSS and was accompanied with edema and congestion, but these conditions were significantly improved after FICZ treatment. Histological analysis showed that there were serious defects in colonic epithelium and crypt after DSS treatment in mice, while FICZ treatment significantly improved these lesions. Compared with DSS treatment group, FICZ upregulated the expression of IL-10 in intestinal epithelium and increased the mRNA expression of proinflammatory cytokines after treatment with DSS, while the expression of inflammatory factors decreased significantly after FICZ treatment. Caco2 cells were cultured in vitro, treated with LPS for 24 hours or co-treated with LPS and FICZ for 24 hours. LPs upregulated mRNA expression of IL-1 尾 and IL-6, but FICZ significantly down-regulated the expression of IL-1 尾 and IL-6. FICZ significantly promoted the expression of IL-10 at protein level and mRNA level. 4. After 24 hours of FICZ treatment, the expression of STAT3 in Caco-2 cells was almost unchanged, but the expression of P-STAT3 was significantly up-regulated. Conclusion 1.FICZ activated AhR significantly alleviated DSS induced colitis in mice. 2. FICZ activated AhR could decrease the expression of inflammatory factors in both animal and cell experiments. Activated AhR can up-regulate the expression of IL-10 and P-STAT3 in intestinal epithelial cells.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R574

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