本文選題：炎癥性腸病 + 纖維化　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：炎癥性腸病(inflammatory bowel disease,IBD)是一種特發(fā)性腸道炎癥性疾病,病變范圍累及回腸、回盲部、結(jié)腸以及直腸。臨床常表現(xiàn)為腹瀉、腹痛、甚至血便,部分患者可有腸梗阻的癥狀。本病主要包括潰瘍性結(jié)腸炎(ulcerative colitis,UC)和克羅恩病(crohn's disease,CD)。UC 是結(jié)腸黏膜層和黏膜下層連續(xù)性炎癥,疾病通常先累及直腸,逐漸向全結(jié)腸蔓延。CD可累及全消化道,為非連續(xù)性全層炎癥,最常累及部位為末端回腸及回盲部,部分累及結(jié)腸和肛周。腸道纖維化是IBD的并發(fā)癥,也是腸腔狹窄導(dǎo)致的腸梗阻和手術(shù)的主要原因,主要病理特點(diǎn)為以膠原為主的細(xì)胞外基質(zhì)(extracellular matrix,ECM)在腸道組織的過度合成以及異常沉積,病程不可逆轉(zhuǎn)。延緩腸道纖維化可改善IBD的自然病程,降低手術(shù)率。作為腸道纖維化的主要效應(yīng)細(xì)胞,腸道成纖維細(xì)胞在炎癥初期的出現(xiàn)是損傷愈合的表現(xiàn),但腸道纖維母細(xì)胞的過度增殖與活化,向肌成纖維細(xì)胞的轉(zhuǎn)化以及分泌大量以膠原為主的ECM和細(xì)胞因子,在腸道纖維化過程中同樣扮演了關(guān)鍵的角色。近來,越來越多的研究證實(shí)內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress,ERS)在一些組織或者細(xì)胞的分化以及凋亡中起到重要的作用,且持續(xù)的ERS和多種器官組織如肝臟、肺、腎以及血管的纖維化有關(guān),是這些器官組織纖維化的機(jī)制之一。但是腸道組織是否存在ERS,以及ERS在腸道纖維母細(xì)胞中的作用,目前鮮有文獻(xiàn)報(bào)道。依據(jù)先前的研究基礎(chǔ),我們推測(cè),IBD的腸道纖維化中,肌纖維母細(xì)胞的ERS在組織纖維化過程中起到重要作用。因此,本研究通過建立三硝基苯磺酸(2,4,6-Trinitrobenzenesulfonic acid,TNBS)誘導(dǎo)的小鼠 IBD纖維化模型以及體外采用衣霉素(tunicamycin,TM)誘導(dǎo)小鼠結(jié)腸纖維母細(xì)胞ERS的方法,在體內(nèi)以及體外觀察小鼠結(jié)腸纖維化組織以及纖維母細(xì)胞ERS標(biāo)志性蛋白,探討腸肌纖維母細(xì)胞ERS在炎癥性腸病腸道纖維化及細(xì)胞重塑過程的作用。具體內(nèi)容概括如下:第一部分ERS相關(guān)蛋白在TNBS誘導(dǎo)的小鼠IBD腸纖維化模型中的動(dòng)態(tài)變化研究目的:ERS相關(guān)蛋白在TNBS誘導(dǎo)的小鼠IBD腸纖維化模型中的動(dòng)態(tài)變化,探討ERS在小鼠UC腸纖維化中的作用。研究方法:雄性C57BL/6小鼠隨機(jī)分為3組,分別為正常組(control組,C組),TNBS組(T組),TNBS+TUDCA組(TT組)。C組給予生理鹽水灌腸作為對(duì)照,T組給予TNBS每周灌腸1次造模,TT組在TNBS灌腸的基礎(chǔ)上,給予ERS保護(hù)劑�；切苋パ跄懰�(tauroursodeoxycholic acid,TUDCA)飼喂。三組動(dòng)物均分別于造模后的第1周,第2周以及第4周隨機(jī)處死,并取結(jié)腸組織進(jìn)行病理切片和蛋白提取處理。采用蘇木精-伊紅染色(HE染色)、馬松(Masson)三聯(lián)染色、免疫組化技術(shù)以及Western blot法觀察結(jié)腸組織的病理染色形態(tài)以及ERS相關(guān)蛋白的表達(dá)。研究結(jié)果:相比于其他兩組,TNBS組的纖維化更為明顯,表現(xiàn)為腸黏膜下層纖維母細(xì)胞大量聚集,后期(灌腸四周后)膠原過度沉積,腸壁結(jié)構(gòu)發(fā)生重塑。同時(shí),ERS現(xiàn)象明顯。在加入ERS調(diào)節(jié)劑TUDCA后,ERS現(xiàn)象減輕,同時(shí)伴有纖維化的明顯緩解。研究結(jié)論:TNBS可以成功誘導(dǎo)小鼠結(jié)腸的纖維化,同時(shí)纖維化的過程有ERS的參與。通過調(diào)節(jié)ERS,可以顯著改善纖維化進(jìn)程。第二部分ERS對(duì)小鼠結(jié)腸纖維母細(xì)胞表型變化的調(diào)節(jié)以及TUDCA對(duì)纖維細(xì)胞母ERS影響的體外研究研究目的:建立TM誘導(dǎo)的小鼠結(jié)腸纖維母細(xì)胞ERS體外模型,并在此基礎(chǔ)研究TUDCA在細(xì)胞層面上對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激的調(diào)控作用。研究方法:本部分實(shí)驗(yàn)分兩個(gè)部分。第一部分是提取原代小鼠結(jié)腸肌纖維母細(xì)胞,6周齡C57BL/6小鼠結(jié)腸采用組織塊貼壁法分離獲取纖維母細(xì)胞,并采用差速貼壁法進(jìn)行細(xì)胞的提純。第二部分是細(xì)胞模型的建立,將提取的纖維母細(xì)胞隨機(jī)分為4組,分別為正常組(control組,C組)、TM組、TUDCA組、TM+TUDCA組,并通過CCK-8細(xì)胞毒力試驗(yàn)確定最適給藥濃度。采用RT-PCR技術(shù)檢測(cè)細(xì)胞中GRP78,CHOP以及α-SMA mRNA的表達(dá)變化,Western blot檢測(cè)相應(yīng)蛋白的表達(dá)情況。研究結(jié)果:TM可導(dǎo)致結(jié)腸肌纖維母細(xì)胞ERS,發(fā)生ERS的纖維母細(xì)胞α-SMA的表達(dá)量明顯升高,提示細(xì)胞發(fā)生重塑,纖維母細(xì)胞轉(zhuǎn)化為肌成纖維細(xì)胞。ERS調(diào)節(jié)劑TUDCA可緩解ERS,并顯著改善纖維母細(xì)胞向肌成纖維細(xì)胞的轉(zhuǎn)化。研究結(jié)論:ERS導(dǎo)致纖維母細(xì)胞向肌成纖維細(xì)胞轉(zhuǎn)化是纖維化的重要機(jī)制,且調(diào)節(jié)ERS可以顯著改善這一進(jìn)程。
[Abstract]:Inflammatory bowel disease (IBD) is an idiopathic intestinal inflammatory disease involving ileum, ileocecal, colon, and rectum. The clinical manifestations are diarrhea, abdominal pain, and even bloody stool. Some patients have symptoms of intestinal obstruction. This disease mainly includes ulcerative colitis (UC) and Crohn's disease (c). Rohn's disease, CD).UC is a continuous inflammation of the mucosa and submucosa of the colon. The disease usually involves the rectum, gradually spreading to the whole colon, and gradually spreading to the whole colon, which can involve the whole digestive tract. It is a discontinuous full layer of inflammation. The most often involved parts are the terminal ileum and the ileocecal part, part of the colon and the perianal. The intestinal fibrosis is the complication of IBD and the narrow of the intestinal cavity. The main reason for the narrow intestinal obstruction and operation is the main pathological feature of the collagen based extracellular matrix (extracellular matrix, ECM) in the intestinal tissue and abnormal deposition. The course of the disease is irreversible. Delayed intestinal fibrosis can improve the natural course of IBD and reduce the rate of operation. As the main effect of intestinal fibrosis, the main effect of intestinal fibrosis is fine. The appearance of intestinal fibroblasts in the early stage of inflammation is the manifestation of damage healing, but the excessive proliferation and activation of the intestinal fibroblasts, the transformation of the fibroblasts to the myofibroblast and the secretion of a large number of collagen based ECM and cytokines also play a key role in the process of intestinal fibrosis. Recently, more and more evidence has been studied. Endoplasmic reticulum stress (ERS) plays an important role in the differentiation and apoptosis of some tissues or cells, and the persistent ERS and a variety of organ tissues, such as liver, lung, kidney and vascular fibrosis, are one of the mechanisms of fibrosis in these organs. But whether there is ERS in the intestinal tissue and ERS, and ERS There are few reports on the role of IBD in intestinal fibroblasts. Based on previous studies, we speculate that the ERS of myofibroblast plays an important role in tissue fibrosis in the intestinal fibrosis of IBD. Therefore, this study was induced by the establishment of three nitrobenzene sulfonic acid (2,4,6-Trinitrobenzenesulfonic acid, TNBS). The rat IBD fibrosis model and the method of using tunicamycin (TM) in vitro to induce the ERS of the colonic fibroblast in mice. In vivo and in vitro, the colon fibrosis tissue and the ERS marker protein of the fibroblast were observed in the mice. The effect of intestinal myofibroblast ERS on intestinal fibrosis and cell remodeling in inflammatory bowel disease was investigated. The specific contents are summarized as follows: the dynamic changes of the ERS related protein in the mouse IBD intestinal fibrosis model induced by TNBS in the first part: the dynamic changes of the ERS related protein in the TNBS induced mouse IBD intestinal fibrosis model, and to explore the role of ERS in the mouse UC intestinal fibrosis. Methods: the male C57BL/6 mice were randomly divided into 3 groups. The normal group (group control, group C), group TNBS (group T), group TNBS+TUDCA (group TT).C group was given saline enema as control, T group was given TNBS weekly enema 1 times, and TT group was fed with ERS protectant tauroxiursodeoxycholic acid on the basis of TNBS enema. The three groups were respectively first after the model. Weeks, second weeks and fourth weeks were executed randomly, and colonic tissue was taken for pathological section and protein extraction. The pathological staining of the colon tissue and the expression of ERS related protein were observed by hematoxylin eosin staining (HE staining), Masson (Masson) triple staining, immunohistochemical technique and Western blot method. In the two group, the fibrosis in the TNBS group was more obvious, showing a large accumulation of fibroblasts in the lower intestinal mucosa, the excessive deposition of collagen and the remodeling of the intestinal wall in the later period (after the enema). At the same time, the phenomenon of ERS was obvious. After the addition of the ERS regulator TUDCA, the ERS phenomenon was relieved and accompanied with the obvious remission of the fibrosis. Conclusion: TNBS can be successfully induced. The fibrosis of colon in mice and the involvement of ERS in the process of fibrosis. Through regulating ERS, the fibrosis process can be significantly improved. Second the regulation of the phenotypic changes of the colonic fibroblasts in mice and the effect of TUDCA on the ERS of the fibroblast mother in vitro: to establish a TM induced ERS body in the colonic fibroblast of mice. External model, and on this basis, we study the regulation of TUDCA on endoplasmic reticulum stress at the cellular level. Research methods: this part of the experiment is divided into two parts. The first part is to extract the primary mouse colonic myofibroblast, and the colon of the 6 week old mice was separated by the tissue block adherence method to obtain the fibroblast, and the differential adherence method was used. Purification of cells. The second part was the establishment of cell model. The extracted fibroblasts were randomly divided into 4 groups: normal group (group control, group C), group TM, group TUDCA, TM+TUDCA group, and the optimum concentration was determined by CCK-8 cell toxicity test. RT-PCR technique was used to detect the expression of GRP78, CHOP, and alpha -SMA mRNA. N blot detected the expression of the corresponding protein. The results of the study showed that TM could lead to the ERS of colonic myofibroblast, the expression of alpha -SMA in ERS fibroblasts increased significantly, suggesting the cell remodeling and the transformation of fibroblast into myofibroblast.ERS modulator TUDCA to relieve ERS and significantly improve fibroblast to myofibroblast. Conclusion: ERS induced fibroblast to myofibroblast transformation is an important mechanism of fibrosis, and regulation of ERS can significantly improve this process.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R574

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Emilien Loeuillard;Julien Bertrand;Anni Herranen;Chloé Melchior;Charlène Guérin;Mo?se Co?ffier;Moutaz Aziz;Pierre Déchelotte;Guillaume Savoye;Rachel Marion-Letellier;;2,4,6-trinitrobenzene sulfonic acid-induced chronic colitis with fibrosis and modulation of TGF-β1 signaling[J];World Journal of Gastroenterology;2014年48期

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本文編號(hào)：1894300