低蛋氨酸对Caco-2肠上皮细胞紧密连接蛋白表达和功能的影响

发布时间：2018-05-14 13:54

本文选题：低蛋氨酸 + 炎症性肠病　；参考：《浙江大学》2017年硕士论文

【摘要】：炎症性肠病(inflammatory bowel disease,IBD)是一组慢性非特异性肠道炎症性疾病,常见为克罗恩病(CD)与溃疡性结肠炎(UC),部分为炎症性肠病未定型(IBDU)。IBD病因及发病机制尚未明确,可能与遗传、环境、微生物感染以及免疫反应等多种因素有关。近年来,肠黏膜屏障功能在IBD发生发展中的作用受到了广泛研究。肠黏膜屏障功能受损既是IBD疾病的表现,又是IBD潜在的致病因素,同时增加IBD复发的风险。所以,维持和恢复肠黏膜屏障功能将有利于提高肠黏膜的防御功能,促进肠黏膜修复有助于维持缓解、减少IBD复发。研究表明,限制蛋氨酸含量能够增强上皮细胞屏障功能。低蛋氨酸处理的LLC-PK1上皮细胞和给予低蛋氨酸饮食的SD大鼠细胞旁路通透性降低,细胞以及大鼠屏障功能增加,紧密连接蛋白的组成和表达发生变化。另外,项目组前期动物实验发现,低蛋氨酸饮食显著降低正常大鼠肠道通透性,并能减轻IBD大鼠肠黏膜炎症损伤,通过改变紧密连接蛋白的表达和功能而增强正常大鼠和IBD大鼠的肠黏膜屏障功能,促进肠黏膜损伤的修复。然而,低蛋氨酸对于受损肠屏障的保护机制并不清楚。由此,我们进一步通过Caco-2肠上皮细胞模型对上述结论进行验证,并检测相关信号通路,初步探索其可能的机制。目的:在动物实验的基础上,进一步研究低蛋氨酸对Caco-2肠上皮细胞及其紧密连接损伤体外模型的影响,并分别检测MLCK-P-MLC以及Rho/ROCK信号通路,初步探索在炎症状态下,低蛋氨酸对紧密连接蛋白表达和功能影响的可能的调控机制。方法:建立Caco-2肠上皮细胞模型,并通过形态学观察、单层细胞完整性及旁路通透性检测、细胞极性研究四个方面联合评价Caco-2模型的可靠性和有效性。根据是否使用低蛋氨酸培养基以及是否经过TNF-α处理48h,将Caco-2细胞分为四组:AA组(对照组)、MR组(低蛋氨酸培养组)、AA+TNF组(正常培养条件下TNF-α处理48h组)、MR+TNF组(低蛋氨酸培养条件下TNF-α处理48h组)。分别检测各组细胞的跨上皮细胞电阻(transepithelial electrical resistance,TEER)、荧光黄(lucifer yellow,LY)透过率和碱性磷酸酶(alkaline phosphatase,AKP)活性;应用透射电镜观察Caco-2单层细胞紧密连接以及微绒毛结构的改变;通过qPCR、Western blot以及免疫荧光等方法,定位、定量检测紧密连接蛋白Claudin-1、Occludin 和 ZO-1;Western blot 实验分别检测 MLCK-P-MLC 信号通路、Rho/ROCK信号通路各相关蛋白的相对表达量,从蛋白水平分析炎症状态下低蛋氨酸对紧密连接蛋白表达和功能影响的可能的调控机制。结果:(1)Caco-2肠上皮细胞体外模型建立成功,具有很好的重复性和可靠性。(2)加入TNF-α培养48h之后,Caco-2肠上皮细胞TEER值显著降低(P0.01),荧光黄透过率显著增高(P0.01);MR组与AA组相比,TEER值显著升高(P0.01),细胞极性显著增加(P0.05),但荧光黄透过率没有发生显著改变;MR+TNF组与AA+TNF组相比,TEER值显著增加且荧光黄透过率显著降低(P0.05)。(3)透射电镜的结果表明,不论正常培养还是低蛋氨酸处理的Caco-2肠上皮细胞,紧密连接结构均位于细胞膜外侧顶端,呈致密带状结构,微绒毛排列紧密、整齐;TNF-α诱导损伤后,肠上皮细胞紧密连接结构破坏、断裂,微绒毛脱落、稀疏、排列紊乱,可见局部微绒毛消失;低蛋氨酸能够减轻TNF-α诱导的紧密连接损伤,减少微绒毛的脱落。(4)qPCR和Western blot的结果表明,各组细胞紧密连接蛋白Claudin-1、Occludin和ZO-1的mRNA和蛋白相对表达量没有显著性差异。(5)免疫荧光结果显示,AA组和MR组Claudin-1、Occludin和ZO-1蛋白沿细胞膜分布,呈蜂巢状线性荧光,TNF-α能诱导紧密连接蛋白异常分布,可见不连续的线性荧光染色,细胞间出现间隙,环断裂,甚至崩解,但MR+TNF组紧密连接的结构和分布变化较AA+TNF组明显好转。(6)Western blot实验对MLCK-P-MLC信号通路的检测结果显示,细胞经TNF-α刺激后,MLCK与pMLC蛋白水平显著增加(P0.05),说明MLCK-P-MLC信号通路被激活;MR+TNF组与AA+TNF组相比,pMLC以及MLCK蛋白表达量显著降低(P0.05),即低蛋氨酸环境能够减弱TNF-α对MLCK-P-MLC信号通路的激活作用。(7)Western blot实验对Rho/ROCK信号通路的检测结果显示,细胞经TNF-α刺激后,p-MYPT1、ROCK1以及ROCK2蛋白水平显著增加(P0.05),说明Rho/ROCK信号通路被激活;MR+TNF组与AA+TNF组相比,p-MYPT1、ROCK1以及ROCK2蛋白相对表达量没有显著性差异。结论:(1)Caco-2肠上皮细胞体外模型建立成功,通过联合评价的方法从形态学观察、TEER值测定、荧光黄透过率测量以及碱性磷酸酶活力测定四个方面来综合评价Caco-2细胞模型的建立,本模型具有很好的重复性和可靠性。(2)TNF-α能够改变紧密连接蛋白的结构和空间分布,使得细胞旁路通透性增加,肠上皮细胞屏障功能受损。(3)低蛋氨酸能够增强肠上皮屏障功能,并且能够改善TNF-α诱导的肠上皮细胞屏障损伤,这种保护作用可能主要是通过改变紧密连接蛋白的结构和空间分布,而对紧密连接蛋白的表达没有影响。(4)TNF-α能够激活MLCK-P-MLC信号通路以及Rho/ROCK信号通路,而低蛋氨酸对肠上皮屏障功能的保护作用可能由于低蛋氨酸环境能够减弱TNF-α对MLCK-P-MLC信号通路的激活作用。
[Abstract]:Inflammatory bowel disease (IBD) is a group of chronic nonspecific intestinal inflammatory diseases, common in Crohn's disease (CD) and ulcerative colitis (UC), partly for the etiology and pathogenesis of inflammatory bowel disease (IBDU).IBD, which may be associated with a variety of factors such as heredity, environment, microbial infection, and immune response. In recent years, the role of intestinal mucosal barrier function in the development of IBD has been widely studied. The impairment of intestinal mucosal barrier function is not only a manifestation of IBD disease, but also a potential pathogenic factor of IBD, which also increases the risk of recurrence of IBD. Therefore, maintaining and restoring the intestinal mucosal barrier function will help to improve the defensive function of the intestinal mucosa and promote the intestinal mucous membrane. Membrane repair helps to maintain remission and reduce the recurrence of IBD. Studies have shown that the restriction of methionine content can enhance the barrier function of epithelial cells. Low methionine treated LLC-PK1 epithelial cells and SD rats giving low methionine diet decrease the bypass permeability, increase the barrier function of cells and rats, and the composition and expression of close connexin In addition, in the early stage of the animal experiment, it was found that the low methionine diet significantly reduced the intestinal permeability of normal rats and alleviated the intestinal mucosal inflammation in IBD rats. By changing the expression and function of the close connexin, the intestinal mucous membrane barrier function of normal rats and IBD rats was enhanced and the repair of intestinal mucosa injury was promoted. However, The protective mechanism of low methionine for impaired intestinal barrier is not clear. Therefore, we further verify the above conclusions through the Caco-2 intestinal epithelial cell model, and detect the related signaling pathways and explore the possible mechanism. Objective: on the basis of animal experiments, we further study the low methionine on the Caco-2 intestinal epithelial cells and their tightens. The effects of MLCK-P-MLC and Rho/ROCK signaling pathway were detected by dense connection, and the possible regulatory mechanism of the effect of low methionine on the expression and function of close connexin in the inflammatory state was preliminarily explored. Methods: the Caco-2 intestinal epithelial cell model was established, and the morphological observation, the integrity of the monolayer cell and the paracrine were observed. The four aspects of permeability test and cell polarity study were combined to evaluate the reliability and effectiveness of the Caco-2 model. The Caco-2 cells were divided into four groups according to whether the use of low methionine medium and the treatment of 48h by TNF- alpha: AA group (control group), MR group (low methionine culture group), AA+TNF group (TNF- alpha treatment 48h group under normal culture), MR+TNF group (TNF- alpha treatment in 48h group under low methionine Culture). The cross epithelial cell resistance (transepithelial electrical resistance, TEER), the transmittance of fluorescent yellow (Lucifer yellow, LY) and alkaline phosphatase (alkaline phosphatase, AKP) activity, respectively, were detected respectively in each group. The close connection and microvilli of the monolayer cells were observed by transmission electron microscopy. Changes in structure; localization and quantitative detection of close connexin Claudin-1, Occludin and ZO-1 by qPCR, Western blot and immunofluorescence. The Western blot test detected the MLCK-P-MLC signal pathway, the relative expression of the related proteins in the Rho/ROCK signaling pathway, and the analysis of the low methionine in the inflammatory state from the protein level to the protein level. The possible regulatory mechanism of the effect of connexin expression and function. Results: (1) Caco-2 intestinal epithelial cells in vitro model was successfully established and had good reproducibility and reliability. (2) after adding TNF- a to 48h, the TEER value of the intestinal epithelial cells of Caco-2 was significantly decreased (P0.01), and the transmittance of fluorescein yellow was significantly increased (P0.01); MR group compared with the AA group, the TEER value was significant. In P0.01, the cell polarity was significantly increased (P0.05), but the transmittance of the fluorescent yellow was not significantly changed. The TEER value of the MR+TNF group was significantly increased and the fluorescence yellow transmittance was significantly decreased (P0.05). (3) the transmission electron microscope showed that the Caco-2 intestinal epithelial cells, both normal and low methionine treatment, were closely connected to the structure of Caco-2. It is located at the top of the outer membrane of the cell membrane, which is dense and banded, and the microvilli are arranged closely and neatly. After TNF- a induced injury, the intestinal epithelial cells closely connect the structure to destroy, break, the microvilli fall off, sparsely, and disarrange, and the local microvilli disappear, and the low methionine can reduce the close connection injury induced by TNF- A and decrease the drop of microvilli. ( 4) the results of qPCR and Western blot showed that the cell close connexin Claudin-1 and the relative expression of mRNA and protein in Occludin and ZO-1 were not significantly different. (5) the immunofluorescence results showed that Claudin-1, Occludin and ZO-1 proteins were distributed along the cell membrane in AA and MR groups, and they showed a honeycomb like linear fluorescence. Often distributed, discontinuous linear fluorescence staining, intercellular space, ring fracture, and even disintegration were found, but the structure and distribution of close connections in the MR+TNF group were obviously better than those in the AA+TNF group. (6) the results of the Western blot test on the MLCK-P-MLC signaling pathway showed that the level of MLCK and pMLC protein increased significantly after the cells were stimulated by TNF- alpha (P0.05) The MLCK-P-MLC signaling pathway was activated, and the expression of pMLC and MLCK protein in group MR+TNF was significantly lower than that in AA+TNF group (P0.05). That is, low methionine environment could weaken the activation of MLCK-P-MLC signaling pathway. (7) Western blot experiments on Rho/ROCK signaling pathway showed that cells were stimulated by TNF- alpha. The level of K1 and ROCK2 protein increased significantly (P0.05), indicating that the Rho/ROCK signaling pathway was activated, and there was no significant difference in the relative expression of p-MYPT1, ROCK1 and ROCK2 protein in MR+TNF group compared with the AA+TNF group. Conclusion: (1) Caco-2 in vitro model of intestinal epithelial cells was established successfully, through joint evaluation methods from morphological observation, TEER determination, fluorescence. The Caco-2 cell model was synthetically evaluated by the four aspects of the measurement of yellow transmittance and the determination of alkaline phosphatase activity. This model has good reproducibility and reliability. (2) TNF- alpha can change the structure and spatial distribution of the close connexin, increase the permeability of the cell bypass, and damage the barrier function of the intestinal epithelial cells. (3) low methionine It can enhance the intestinal epithelial barrier function, and can improve the intestinal epithelial barrier damage induced by TNF- alpha. This protective effect may be mainly by changing the structure and spatial distribution of tight connexin, but not affecting the expression of close connexin. (4) TNF- alpha can activate the MLCK-P-MLC signaling pathway and the Rho/ROCK signaling pathway. The protective effect of low methionine on intestinal epithelial barrier function may be due to the low methionine environment which can weaken the activation of TNF- alpha on MLCK-P-MLC signaling pathway.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574.62

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