本文選題：同型半胱氨酸　切入點(diǎn)：炎癥性腸病　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:炎癥性腸病(Inflammatory bowel disease,IBD)是一種目前發(fā)病機(jī)制尚不十分明確的慢性腸道炎癥性疾病。既往研究表明,腸道粘膜緊密連接結(jié)構(gòu)的破壞引起腸粘膜通透性增加對(duì)IBD的發(fā)病過(guò)程具有重要影響。同型半胱氨酸(homocysteine,Hcy)是一種含硫氨基酸,是甲硫氨酸代謝的重要中間產(chǎn)物。研究發(fā)現(xiàn)Hcy可能通過(guò)多種途徑影響內(nèi)皮細(xì)胞屏障功能,提高內(nèi)皮細(xì)胞通透性,促進(jìn)炎癥反應(yīng)。炎癥性腸病(IBD)患者血漿和結(jié)腸粘膜組織中Hcy水平增高,但Hcy是否通過(guò)影響腸上皮細(xì)胞緊密連接結(jié)構(gòu)及功能,進(jìn)一步影響腸粘膜通透性參與IBD的病理生理過(guò)程尚不明確。因此,本研究擬在建立大鼠TNBS/乙醇結(jié)腸炎模型及人結(jié)腸癌Caco-2細(xì)胞株的基礎(chǔ)上,探討同型半胱氨酸對(duì)實(shí)驗(yàn)性結(jié)腸炎腸粘膜通透性及Caco-2細(xì)胞單層通透性的影響及其作用機(jī)理。目的:探討同型半胱氨酸(Hcy)對(duì)實(shí)驗(yàn)性結(jié)腸炎大鼠小腸粘膜通透性及Caco-2細(xì)胞單層通透性的影響及作用機(jī)理。方法:1.Caco-2細(xì)胞經(jīng)Hcy預(yù)處理不同信號(hào)通路抑制劑(ERK抑制劑PD98059,P38MAPK抑制劑SB203580,Rho抑制劑Y27632,Ca~(2+)/Calmodulin抑制劑KN62,PKC抑制劑Staurosporine以及MLCK抑制劑ML-7)之后,加入FITC。記錄Caco-2細(xì)胞TEER隨時(shí)間變化情況,同時(shí)于lower chamber取樣,樣品中FITC濃度通過(guò)熒光分光光度計(jì)測(cè)定。實(shí)驗(yàn)結(jié)束后收集細(xì)胞懸液,Western blot方法檢測(cè)細(xì)胞中MEK,ERK,p-ERK,MLCK,p-MLCK,ZO-1,Claudin-1,Claudin-2,Occludin蛋白表達(dá)。2.使用2,4,6-三硝基苯磺酸(TNBS)/乙醇灌腸制備實(shí)驗(yàn)性大鼠結(jié)腸炎模型,采用皮下注射同型半胱氨酸造成高同型半胱氨酸血癥(HHcy)模型。3.SD雄性大鼠,分成4組:A組為對(duì)照組(NS注射+NS灌腸),B組為對(duì)照+Hcy注射組(Hcy注射+NS灌腸),C組為TNBS模型組(NS注射+TNBS/乙醇灌腸),D組為TNBS模型+Hcy注射組(Hcy皮注射+TNBS/乙醇灌腸)。4.記錄觀察大鼠每日體重變化及糞便情況,根據(jù)大鼠癥狀及體重變化情況進(jìn)行疾病活動(dòng)性評(píng)分(DAI),切取實(shí)驗(yàn)性結(jié)腸炎大鼠結(jié)腸標(biāo)本行HE染色,并行結(jié)腸組織學(xué)損傷評(píng)分(HI)5.大鼠結(jié)腸Hcy及MLCK水平通過(guò)ELISA方法檢測(cè),大鼠結(jié)腸MPO活性通過(guò)四甲基聯(lián)苯胺法測(cè)定,大鼠小腸粘膜組織中MEK,ERK,p-ERK,MLCK,p-MLCK,ZO-1,Claudin-1,Claudin-2,Occludin蛋白表達(dá)使用免疫組化及western blot方法測(cè)定,MLCK m RNA表達(dá)通過(guò)RT-q PCR方法檢測(cè)。結(jié)果:與對(duì)照組相比,Caco-2細(xì)胞使用50μmol/L Hcy預(yù)處理時(shí)TEER降低明顯。與Hcy組相比,Caco-2細(xì)胞經(jīng)ML-7及PD98059藥物預(yù)處理后TEER下降減緩,FITC跨膜轉(zhuǎn)運(yùn)量有所降低,MLCK,p-MLCK,Claudin-2蛋白表達(dá)有所降低,ZO-1,Claudin-1,Occludin蛋白表達(dá)有所回升,表明MEK-ERK-MLCK信號(hào)通路在Hcy影響Caco-2細(xì)胞通透性的過(guò)程中具有影響。與正常對(duì)照組及TNBS模型對(duì)照組相比,TNBS模型+Hcy皮下注射組DAI,HI,MPO顯著升高,結(jié)腸MLCK水平明顯增加,大鼠小腸組織中MEK,ERK,p-ERK,MLCK,p-MLCK,Claudin-2蛋白表達(dá)升高,ZO-1,Claudin-1,Occludin蛋白降低(P0.05),表明Hcy可能通過(guò)調(diào)控MEK-ERK-MLCK通路影響實(shí)驗(yàn)性結(jié)腸炎大鼠小腸粘膜細(xì)胞緊密連接蛋白表達(dá),增加腸粘膜通透性。結(jié)論:Hcy可影響Caco-2細(xì)胞及實(shí)驗(yàn)性結(jié)腸炎大鼠小腸粘膜通透性,機(jī)制可能與調(diào)控MLCK表達(dá)影響腸道粘膜上皮細(xì)胞緊密連接通透性有關(guān)。
[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.

【學(xué)位授予單位】：安徽醫(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

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本文編號(hào)：1713706