本文選題：低蛋氨酸 + 炎癥性腸病　； 參考：《浙江大學》2017年碩士論文

【摘要】：炎癥性腸病(inflammatory bowel disease,IBD)是一組慢性非特異性腸道炎癥性疾病,常見為克羅恩病(CD)與潰瘍性結(jié)腸炎(UC),部分為炎癥性腸病未定型(IBDU)。IBD病因及發(fā)病機制尚未明確,可能與遺傳、環(huán)境、微生物感染以及免疫反應等多種因素有關。近年來,腸黏膜屏障功能在IBD發(fā)生發(fā)展中的作用受到了廣泛研究。腸黏膜屏障功能受損既是IBD疾病的表現(xiàn),又是IBD潛在的致病因素,同時增加IBD復發(fā)的風險。所以,維持和恢復腸黏膜屏障功能將有利于提高腸黏膜的防御功能,促進腸黏膜修復有助于維持緩解、減少IBD復發(fā)。研究表明,限制蛋氨酸含量能夠增強上皮細胞屏障功能。低蛋氨酸處理的LLC-PK1上皮細胞和給予低蛋氨酸飲食的SD大鼠細胞旁路通透性降低,細胞以及大鼠屏障功能增加,緊密連接蛋白的組成和表達發(fā)生變化。另外,項目組前期動物實驗發(fā)現(xiàn),低蛋氨酸飲食顯著降低正常大鼠腸道通透性,并能減輕IBD大鼠腸黏膜炎癥損傷,通過改變緊密連接蛋白的表達和功能而增強正常大鼠和IBD大鼠的腸黏膜屏障功能,促進腸黏膜損傷的修復。然而,低蛋氨酸對于受損腸屏障的保護機制并不清楚。由此,我們進一步通過Caco-2腸上皮細胞模型對上述結(jié)論進行驗證,并檢測相關信號通路,初步探索其可能的機制。目的:在動物實驗的基礎上,進一步研究低蛋氨酸對Caco-2腸上皮細胞及其緊密連接損傷體外模型的影響,并分別檢測MLCK-P-MLC以及Rho/ROCK信號通路,初步探索在炎癥狀態(tài)下,低蛋氨酸對緊密連接蛋白表達和功能影響的可能的調(diào)控機制。方法:建立Caco-2腸上皮細胞模型,并通過形態(tài)學觀察、單層細胞完整性及旁路通透性檢測、細胞極性研究四個方面聯(lián)合評價Caco-2模型的可靠性和有效性。根據(jù)是否使用低蛋氨酸培養(yǎng)基以及是否經(jīng)過TNF-α處理48h,將Caco-2細胞分為四組:AA組(對照組)、MR組(低蛋氨酸培養(yǎng)組)、AA+TNF組(正常培養(yǎng)條件下TNF-α處理48h組)、MR+TNF組(低蛋氨酸培養(yǎng)條件下TNF-α處理48h組)。分別檢測各組細胞的跨上皮細胞電阻(transepithelial electrical resistance,TEER)、熒光黃(lucifer yellow,LY)透過率和堿性磷酸酶(alkaline phosphatase,AKP)活性;應用透射電鏡觀察Caco-2單層細胞緊密連接以及微絨毛結(jié)構(gòu)的改變;通過qPCR、Western blot以及免疫熒光等方法,定位、定量檢測緊密連接蛋白Claudin-1、Occludin 和 ZO-1;Western blot 實驗分別檢測 MLCK-P-MLC 信號通路、Rho/ROCK信號通路各相關蛋白的相對表達量,從蛋白水平分析炎癥狀態(tài)下低蛋氨酸對緊密連接蛋白表達和功能影響的可能的調(diào)控機制。結(jié)果:(1)Caco-2腸上皮細胞體外模型建立成功,具有很好的重復性和可靠性。(2)加入TNF-α培養(yǎng)48h之后,Caco-2腸上皮細胞TEER值顯著降低(P0.01),熒光黃透過率顯著增高(P0.01);MR組與AA組相比,TEER值顯著升高(P0.01),細胞極性顯著增加(P0.05),但熒光黃透過率沒有發(fā)生顯著改變;MR+TNF組與AA+TNF組相比,TEER值顯著增加且熒光黃透過率顯著降低(P0.05)。(3)透射電鏡的結(jié)果表明,不論正常培養(yǎng)還是低蛋氨酸處理的Caco-2腸上皮細胞,緊密連接結(jié)構(gòu)均位于細胞膜外側(cè)頂端,呈致密帶狀結(jié)構(gòu),微絨毛排列緊密、整齊;TNF-α誘導損傷后,腸上皮細胞緊密連接結(jié)構(gòu)破壞、斷裂,微絨毛脫落、稀疏、排列紊亂,可見局部微絨毛消失;低蛋氨酸能夠減輕TNF-α誘導的緊密連接損傷,減少微絨毛的脫落。(4)qPCR和Western blot的結(jié)果表明,各組細胞緊密連接蛋白Claudin-1、Occludin和ZO-1的mRNA和蛋白相對表達量沒有顯著性差異。(5)免疫熒光結(jié)果顯示,AA組和MR組Claudin-1、Occludin和ZO-1蛋白沿細胞膜分布,呈蜂巢狀線性熒光,TNF-α能誘導緊密連接蛋白異常分布,可見不連續(xù)的線性熒光染色,細胞間出現(xiàn)間隙,環(huán)斷裂,甚至崩解,但MR+TNF組緊密連接的結(jié)構(gòu)和分布變化較AA+TNF組明顯好轉(zhuǎn)。(6)Western blot實驗對MLCK-P-MLC信號通路的檢測結(jié)果顯示,細胞經(jīng)TNF-α刺激后,MLCK與pMLC蛋白水平顯著增加(P0.05),說明MLCK-P-MLC信號通路被激活;MR+TNF組與AA+TNF組相比,pMLC以及MLCK蛋白表達量顯著降低(P0.05),即低蛋氨酸環(huán)境能夠減弱TNF-α對MLCK-P-MLC信號通路的激活作用。(7)Western blot實驗對Rho/ROCK信號通路的檢測結(jié)果顯示,細胞經(jīng)TNF-α刺激后,p-MYPT1、ROCK1以及ROCK2蛋白水平顯著增加(P0.05),說明Rho/ROCK信號通路被激活;MR+TNF組與AA+TNF組相比,p-MYPT1、ROCK1以及ROCK2蛋白相對表達量沒有顯著性差異。結(jié)論:(1)Caco-2腸上皮細胞體外模型建立成功,通過聯(lián)合評價的方法從形態(tài)學觀察、TEER值測定、熒光黃透過率測量以及堿性磷酸酶活力測定四個方面來綜合評價Caco-2細胞模型的建立,本模型具有很好的重復性和可靠性。(2)TNF-α能夠改變緊密連接蛋白的結(jié)構(gòu)和空間分布,使得細胞旁路通透性增加,腸上皮細胞屏障功能受損。(3)低蛋氨酸能夠增強腸上皮屏障功能,并且能夠改善TNF-α誘導的腸上皮細胞屏障損傷,這種保護作用可能主要是通過改變緊密連接蛋白的結(jié)構(gòu)和空間分布,而對緊密連接蛋白的表達沒有影響。(4)TNF-α能夠激活MLCK-P-MLC信號通路以及Rho/ROCK信號通路,而低蛋氨酸對腸上皮屏障功能的保護作用可能由于低蛋氨酸環(huán)境能夠減弱TNF-α對MLCK-P-MLC信號通路的激活作用。
[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.

【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R574.62

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