肠肌纤维母细胞内质网应激在炎症性肠病肠道纤维化及细胞重塑过程的作用

发布时间：2018-05-15 22:43

本文选题：炎症性肠病 + 纤维化　；参考：《南方医科大学》2017年硕士论文

【摘要】：炎症性肠病(inflammatory bowel disease,IBD)是一种特发性肠道炎症性疾病,病变范围累及回肠、回盲部、结肠以及直肠。临床常表现为腹泻、腹痛、甚至血便,部分患者可有肠梗阻的症状。本病主要包括溃疡性结肠炎(ulcerative colitis,UC)和克罗恩病(crohn's disease,CD)。UC 是结肠黏膜层和黏膜下层连续性炎症,疾病通常先累及直肠,逐渐向全结肠蔓延。CD可累及全消化道,为非连续性全层炎症,最常累及部位为末端回肠及回盲部,部分累及结肠和肛周。肠道纤维化是IBD的并发症,也是肠腔狭窄导致的肠梗阻和手术的主要原因,主要病理特点为以胶原为主的细胞外基质(extracellular matrix,ECM)在肠道组织的过度合成以及异常沉积,病程不可逆转。延缓肠道纤维化可改善IBD的自然病程,降低手术率。作为肠道纤维化的主要效应细胞,肠道成纤维细胞在炎症初期的出现是损伤愈合的表现,但肠道纤维母细胞的过度增殖与活化,向肌成纤维细胞的转化以及分泌大量以胶原为主的ECM和细胞因子,在肠道纤维化过程中同样扮演了关键的角色。近来,越来越多的研究证实内质网应激(endoplasmic reticulum stress,ERS)在一些组织或者细胞的分化以及凋亡中起到重要的作用,且持续的ERS和多种器官组织如肝脏、肺、肾以及血管的纤维化有关,是这些器官组织纤维化的机制之一。但是肠道组织是否存在ERS,以及ERS在肠道纤维母细胞中的作用,目前鲜有文献报道。依据先前的研究基础,我们推测,IBD的肠道纤维化中,肌纤维母细胞的ERS在组织纤维化过程中起到重要作用。因此,本研究通过建立三硝基苯磺酸(2,4,6-Trinitrobenzenesulfonic acid,TNBS)诱导的小鼠 IBD纤维化模型以及体外采用衣霉素(tunicamycin,TM)诱导小鼠结肠纤维母细胞ERS的方法,在体内以及体外观察小鼠结肠纤维化组织以及纤维母细胞ERS标志性蛋白,探讨肠肌纤维母细胞ERS在炎症性肠病肠道纤维化及细胞重塑过程的作用。具体内容概括如下:第一部分ERS相关蛋白在TNBS诱导的小鼠IBD肠纤维化模型中的动态变化研究目的:ERS相关蛋白在TNBS诱导的小鼠IBD肠纤维化模型中的动态变化,探讨ERS在小鼠UC肠纤维化中的作用。研究方法:雄性C57BL/6小鼠随机分为3组,分别为正常组(control组,C组),TNBS组(T组),TNBS+TUDCA组(TT组)。C组给予生理盐水灌肠作为对照,T组给予TNBS每周灌肠1次造模,TT组在TNBS灌肠的基础上,给予ERS保护剂牛磺熊去氧胆酸(tauroursodeoxycholic acid,TUDCA)饲喂。三组动物均分别于造模后的第1周,第2周以及第4周随机处死,并取结肠组织进行病理切片和蛋白提取处理。采用苏木精-伊红染色(HE染色)、马松(Masson)三联染色、免疫组化技术以及Western blot法观察结肠组织的病理染色形态以及ERS相关蛋白的表达。研究结果:相比于其他两组,TNBS组的纤维化更为明显,表现为肠黏膜下层纤维母细胞大量聚集,后期(灌肠四周后)胶原过度沉积,肠壁结构发生重塑。同时,ERS现象明显。在加入ERS调节剂TUDCA后,ERS现象减轻,同时伴有纤维化的明显缓解。研究结论:TNBS可以成功诱导小鼠结肠的纤维化,同时纤维化的过程有ERS的参与。通过调节ERS,可以显著改善纤维化进程。第二部分ERS对小鼠结肠纤维母细胞表型变化的调节以及TUDCA对纤维细胞母ERS影响的体外研究研究目的:建立TM诱导的小鼠结肠纤维母细胞ERS体外模型,并在此基础研究TUDCA在细胞层面上对内质网应激的调控作用。研究方法:本部分实验分两个部分。第一部分是提取原代小鼠结肠肌纤维母细胞,6周龄C57BL/6小鼠结肠采用组织块贴壁法分离获取纤维母细胞,并采用差速贴壁法进行细胞的提纯。第二部分是细胞模型的建立,将提取的纤维母细胞随机分为4组,分别为正常组(control组,C组)、TM组、TUDCA组、TM+TUDCA组,并通过CCK-8细胞毒力试验确定最适给药浓度。采用RT-PCR技术检测细胞中GRP78,CHOP以及α-SMA mRNA的表达变化,Western blot检测相应蛋白的表达情况。研究结果:TM可导致结肠肌纤维母细胞ERS,发生ERS的纤维母细胞α-SMA的表达量明显升高,提示细胞发生重塑,纤维母细胞转化为肌成纤维细胞。ERS调节剂TUDCA可缓解ERS,并显著改善纤维母细胞向肌成纤维细胞的转化。研究结论:ERS导致纤维母细胞向肌成纤维细胞转化是纤维化的重要机制,且调节ERS可以显著改善这一进程。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574

【参考文献】