同型半胱氨酸调控MLCK影响实验性结肠炎肠粘膜通透性的作用研究

发布时间：2018-04-05 07:10

本文选题：同型半胱氨酸　切入点：炎症性肠病　出处：《安徽医科大学》2017年硕士论文

【摘要】：背景:炎症性肠病(Inflammatory bowel disease,IBD)是一种目前发病机制尚不十分明确的慢性肠道炎症性疾病。既往研究表明,肠道粘膜紧密连接结构的破坏引起肠粘膜通透性增加对IBD的发病过程具有重要影响。同型半胱氨酸(homocysteine,Hcy)是一种含硫氨基酸,是甲硫氨酸代谢的重要中间产物。研究发现Hcy可能通过多种途径影响内皮细胞屏障功能,提高内皮细胞通透性,促进炎症反应。炎症性肠病(IBD)患者血浆和结肠粘膜组织中Hcy水平增高,但Hcy是否通过影响肠上皮细胞紧密连接结构及功能,进一步影响肠粘膜通透性参与IBD的病理生理过程尚不明确。因此,本研究拟在建立大鼠TNBS/乙醇结肠炎模型及人结肠癌Caco-2细胞株的基础上,探讨同型半胱氨酸对实验性结肠炎肠粘膜通透性及Caco-2细胞单层通透性的影响及其作用机理。目的:探讨同型半胱氨酸(Hcy)对实验性结肠炎大鼠小肠粘膜通透性及Caco-2细胞单层通透性的影响及作用机理。方法:1.Caco-2细胞经Hcy预处理不同信号通路抑制剂(ERK抑制剂PD98059,P38MAPK抑制剂SB203580,Rho抑制剂Y27632,Ca~(2+)/Calmodulin抑制剂KN62,PKC抑制剂Staurosporine以及MLCK抑制剂ML-7)之后,加入FITC。记录Caco-2细胞TEER随时间变化情况,同时于lower chamber取样,样品中FITC浓度通过荧光分光光度计测定。实验结束后收集细胞悬液,Western blot方法检测细胞中MEK,ERK,p-ERK,MLCK,p-MLCK,ZO-1,Claudin-1,Claudin-2,Occludin蛋白表达。2.使用2,4,6-三硝基苯磺酸(TNBS)/乙醇灌肠制备实验性大鼠结肠炎模型,采用皮下注射同型半胱氨酸造成高同型半胱氨酸血症(HHcy)模型。3.SD雄性大鼠,分成4组:A组为对照组(NS注射+NS灌肠),B组为对照+Hcy注射组(Hcy注射+NS灌肠),C组为TNBS模型组(NS注射+TNBS/乙醇灌肠),D组为TNBS模型+Hcy注射组(Hcy皮注射+TNBS/乙醇灌肠)。4.记录观察大鼠每日体重变化及粪便情况,根据大鼠症状及体重变化情况进行疾病活动性评分(DAI),切取实验性结肠炎大鼠结肠标本行HE染色,并行结肠组织学损伤评分(HI)5.大鼠结肠Hcy及MLCK水平通过ELISA方法检测,大鼠结肠MPO活性通过四甲基联苯胺法测定,大鼠小肠粘膜组织中MEK,ERK,p-ERK,MLCK,p-MLCK,ZO-1,Claudin-1,Claudin-2,Occludin蛋白表达使用免疫组化及western blot方法测定,MLCK m RNA表达通过RT-q PCR方法检测。结果:与对照组相比,Caco-2细胞使用50μmol/L Hcy预处理时TEER降低明显。与Hcy组相比,Caco-2细胞经ML-7及PD98059药物预处理后TEER下降减缓,FITC跨膜转运量有所降低,MLCK,p-MLCK,Claudin-2蛋白表达有所降低,ZO-1,Claudin-1,Occludin蛋白表达有所回升,表明MEK-ERK-MLCK信号通路在Hcy影响Caco-2细胞通透性的过程中具有影响。与正常对照组及TNBS模型对照组相比,TNBS模型+Hcy皮下注射组DAI,HI,MPO显著升高,结肠MLCK水平明显增加,大鼠小肠组织中MEK,ERK,p-ERK,MLCK,p-MLCK,Claudin-2蛋白表达升高,ZO-1,Claudin-1,Occludin蛋白降低(P0.05),表明Hcy可能通过调控MEK-ERK-MLCK通路影响实验性结肠炎大鼠小肠粘膜细胞紧密连接蛋白表达,增加肠粘膜通透性。结论:Hcy可影响Caco-2细胞及实验性结肠炎大鼠小肠粘膜通透性,机制可能与调控MLCK表达影响肠道粘膜上皮细胞紧密连接通透性有关。
[Abstract]:Background: inflammatory bowel disease (Inflammatory bowel, disease, IBD) is a kind of the pathogenesis is not very clear of the chronic inflammatory bowel disease. Previous studies showed that intestinal mucosal tight junction damage of intestinal mucosal permeability increased the incidence of IBD has an important influence. Homocysteine (homocysteine, Hcy) is a a sulfur-containing amino acid is an important intermediate product of methionine metabolism. The study found that Hcy may affect endothelial barrier function through a variety of channels, improve the permeability of endothelial cells, promote inflammation. Inflammatory bowel disease (IBD) and increased plasma Hcy levels in patients with colon mucosa, but Hcy is closely connected to the structure and function of the effect of intestinal epithelial cells further, influence the permeability of the intestinal mucosa involved in the pathophysiology of IBD is still not clear. Therefore, this study intends to establish rat colitis TNBS/ ethanol The basic model and human colonic carcinoma cell line Caco-2, to investigate the effect of homocysteine on experimental colitis intestinal permeability and Caco-2 cell monolayer permeability and its mechanism of action. Objective: To investigate the effect of homocysteine (Hcy) effect on rat intestinal mucosal permeability and Caco-2 cell monolayer permeability in experimental colitis and mechanism. Hcy: pretreatment of 1.Caco-2 cells with different signaling pathway inhibitors (ERK inhibitor PD98059, P38MAPK inhibitor SB203580, Rho inhibitor Y27632, Ca~ (2+) /Calmodulin inhibitor KN62, PKC inhibitor Staurosporine and MLCK inhibitor ML-7) after adding FITC. record Caco-2 cell TEER changes in lower chamber and FITC concentration in the samples by sampling. Fluorescence spectrophotometer. After the experiment collected cell suspension, Western method for the detection of blot cells in MEK, ERK, p-ERK, MLCK, P -MLCK, ZO-1, Claudin-1, Claudin-2, Occludin protein expression of.2. using 2,4,6- three trinitrobenzene sulfonic acid (TNBS) / ethanol enema preparation model of experimental colitis in rats by subcutaneous injection of homocysteine caused by hyperhomocysteinemia (HHcy) model of.3.SD male rats were divided into 4 groups: group A control group (NS injection of +NS), enema group B as control group were injected with +Hcy (Hcy +NS, C injection enema) group TNBS model group (NS injection of +TNBS/ ethanol enema group D), TNBS model +Hcy injection group (Hcy percutaneous injection of +TNBS/ ethanol enema).4. rats were observed daily records of weight changes and feces, disease activity the score of rats according to the symptoms and body weight changes (DAI), cut from the experimental colitis rat colon HE staining, colon histological injury score (HI) 5. in rat colon Hcy and MLCK levels detected by the ELISA method, the rat colon MPO activity by four Determination of benzidine by rat small intestinal mucosa in MEK, ERK, p-ERK, MLCK, p-MLCK, ZO-1, Claudin-1, Claudin-2, and Western were determined using immunohistochemical blot method for protein expression of Occludin, MLCK m RNA RT-q expression through PCR assay. Results: compared with the control group, Caco-2 cells using 50 mol/L Hcy pretreatment TEER decreased significantly. Compared with the Hcy group, Caco-2 cells were treated with ML-7 and PD98059 drug pretreatment decreased TEER after slow FITC transmembrane transport decreased, MLCK, p-MLCK, Claudin-2 protein expression decreased, ZO-1, Claudin-1, Occludin protein expression increased, suggesting that MEK-ERK-MLCK signaling has influence in the process of Caco-2 cell permeability effects of Hcy. Compared with the normal control group and TNBS model group, TNBS model of subcutaneous injection of +Hcy group DAI, HI, MPO were significantly increased, MLCK level was significantly increased in the colon, small intestine tissue of rats in MEK, ERK, p-E RK, MLCK, p-MLCK, Claudin-2 protein expression increased, ZO-1, Claudin-1, Occludin protein (P0.05), showed that reduced Hcy may affect the intestinal mucosal cell in rats with experimental colitis control the expression of tight junction protein MEK-ERK-MLCK pathway, intestinal permeability increased. Conclusion: Hcy can affect Caco-2 cells and experimental colitis rat intestinal mucosal permeability and the mechanism might be related to the regulation of the expression of MLCK of intestinal mucosal epithelial tight junction permeability.

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

【参考文献】