發(fā)布時(shí)間：2018-05-14 00:30

  本文選題：促腎上腺皮質(zhì)激素釋放激素 + 腸粘膜屏障��； 參考：《山東大學(xué)》2016年碩士論文

【摘要】：研究背景腸易激綜合征(irritable bowel syndrome, IBS)是指伴有反復(fù)的腹痛和腸道習(xí)慣改變,沒有可解釋癥狀的器質(zhì)性病變存在的慢性功能性消化道疾病,是研究最多的功能性胃腸病(functional gastrointestinal disorders, FGIDs)之一,其發(fā)病可能與精神應(yīng)激和腦腸軸功能紊亂有關(guān)。精神應(yīng)激可以通過破壞腦腸軸,導(dǎo)致腸道屏障的破壞和腸道通透性的增加,從而引起或者加劇腸道疾病的發(fā)生發(fā)展,比如壓力可以加重IBS的癥狀。在應(yīng)激過程中,腦腸軸的一種主要激素促腎上腺皮質(zhì)激素釋放激素(corticotropin releasing factor, CRF)的含量是增加的。腸道屏障包括粘液屏障和上皮屏障,對腸道功能的維持非常重要。杯狀細(xì)胞合成的一些分泌產(chǎn)物是腸道粘膜屏障的重要組成成分。上皮間的緊密連接蛋白是腸道上皮屏障的重要組成成分。LS174T細(xì)胞是人結(jié)腸腺癌細(xì)胞,有杯狀細(xì)胞的表型和特性,能夠合成并分泌粘蛋白和與粘蛋白有關(guān)的其他分泌產(chǎn)物,可以用來研究杯狀細(xì)胞的功能。LS174T細(xì)胞也是一種上皮細(xì)胞,可以用來研究腸道上皮屏障的功能。精神應(yīng)激是腸道屏障破壞的一個(gè)危險(xiǎn)因素,那么精神應(yīng)激如何通過CRF調(diào)節(jié)杯狀細(xì)胞的功能以及上皮間的緊密連接蛋白而影響腸道屏障的具體機(jī)制還尚不清楚,我們猜想,CRF可以通過改變腸道杯狀細(xì)胞的分泌產(chǎn)物和上皮間緊密連接蛋白的表達(dá)從而破壞腸道屏障的功能。目的探討促腎上腺皮質(zhì)激素釋放激素通過調(diào)節(jié)腸道杯狀細(xì)胞的分泌產(chǎn)物和上皮間緊密連接蛋白對腸道屏障的影響。方法1.培養(yǎng)人結(jié)腸腺癌細(xì)胞LS174T細(xì)胞,應(yīng)用不同濃度10-9, 10-10, 10-11, 10-12 mol/L)的促腎上腺皮質(zhì)激素釋放激素(CRF)作用于LS174T細(xì)胞6小時(shí),PBS作為對照組；10-10 mol/L的CRF作用于LS174T細(xì)胞6小時(shí)或24小時(shí),PBS作為對照組。2.利用Real-Time qPCR技術(shù)觀察不同濃度的CRF對LS174T細(xì)胞分泌產(chǎn)物相關(guān)基因MUC2, FUT2和TFF3的mRNA水平表達(dá)量的影響,以確定CRF作用的最佳濃度。3.利用Real-Time qPCR技術(shù)觀察10-10 mol/L的CRF對LS174T細(xì)胞中MUC2, FUT2, TFF3, claudin-1, claudin-2, occludin和ZO-1的mRNA水平表達(dá)量的影響。4.利用Western blot技術(shù)觀察10-10 mol/L的CRF對LS174T細(xì)胞中FUT2,TFF3, claudin-1, claudin-2, occludin和ZO-1的蛋白水平表達(dá)量的影響。5.利用免疫熒光染色和ELISA技術(shù)觀察CRF對LS174T細(xì)胞中MUC2蛋白水平表達(dá)量的影響。結(jié)果1. Real-Time qPCR結(jié)果顯示,與PBS對照組相比,不同濃度的CRF對LS174T細(xì)胞中MUC2, FUT2口TFF3有不同的影響,10-10 mol·L-1的CRF對腸道杯狀細(xì)胞分泌產(chǎn)物的影響相對明顯。2. Real-Time qPCR結(jié)果顯示,10-10 mol·L-1的CRF作用于LS174T細(xì)胞6小時(shí)后,MUC2 (P0.01), FUT2 (P 0.001)和TFF3 (P 0.0001)的mRNA表達(dá)量均下降；CRF作用于LS174T細(xì)胞24小時(shí)后,與對照組相比,MUC2和FUT2的mRNA表達(dá)量無明顯差異(NS),而TFF3的mRNA表達(dá)量仍下降(P0.001)。3.細(xì)胞免疫熒光和ELISA的結(jié)果顯示,與PBS對照組相比,CRF作用于LS174T細(xì)胞6小時(shí)后,MUC2的蛋白表達(dá)量降低(P0.05),而CRF作用于細(xì)胞24小時(shí)后,MUC2的表達(dá)無顯著性差異(NS)。4. Western blot結(jié)果顯示,與PBS對照組相比,CRF作用LS174T細(xì)胞6小時(shí)后,TFF3的蛋白表達(dá)量是下降的(P 0.001), CRF作用24小時(shí)后,FUT2和TFF3的蛋白水平均是下降的(P0.001)。5. Real-Time qPCR結(jié)果顯示,10-10 mol/L CRF作用于LS174T細(xì)胞6小時(shí)后,claudin-2的mRNA表達(dá)水平增加(p0.001),而occludin和ZO-1的mRNA表達(dá)水平在CRF作用24小時(shí)后下降(P0.001)。6. Western blot結(jié)果顯示,CRF作用于LS174T細(xì)胞6小時(shí)后,claudin-2的蛋白表達(dá)量增加(p0.001),而CRF作用24小時(shí)后,occludin和ZO-1的蛋白表達(dá)量下降(P0.001)。7.CRF作用于LS174T細(xì)胞6小時(shí)后,claudin-1的mRNA和蛋白表達(dá)量與PBS對照組相比均無顯著性差異(NS)。結(jié)論1.CRF可以下調(diào)MUC2, FUT2和TFF3的表達(dá)量,導(dǎo)致腸粘膜屏障的組成成分發(fā)生改變,對維持腸粘膜屏障功能不利。2.CRF可以上調(diào)claudin-2,下調(diào)occludin和ZO-1,導(dǎo)致細(xì)胞間緊密連接蛋白的成分發(fā)生改變,對維持腸道上皮屏障的完整性不利。3.CRF對腸道杯狀細(xì)胞分泌產(chǎn)物的分泌以及上皮間緊密連接蛋白的表達(dá)的影響可能是精神應(yīng)激導(dǎo)致腸道屏障功能破壞的原因。
[Abstract]:Background irritable bowel syndrome (IBS) is a chronic functional digestive tract disease associated with recurrent abdominal pain and changes in intestinal habits and no interpretable symptoms of organic disease. It is one of the most studied functional gastrointestinal diseases (functional gastrointestinal disorders, FGIDs), and its pathogenesis may be with sperm. Mental stress is related to the dysfunction of the brain axis. Mental stress can cause or aggravate the development of intestinal diseases by destroying the brain axis, causing the destruction of the intestinal barrier and increasing the permeability of the intestine, such as stress can aggravate the symptoms of IBS. In the process of stress, a major hormone Corticotropin in the brain axis The content of corticotropin releasing factor (CRF) is increased. The intestinal barrier, including the mucous barrier and the epithelial barrier, is very important for the maintenance of intestinal function. Some secretory products of the goblet cell are important components of the intestinal mucosal barrier. The close connexin between the epithelium is an important group of the intestinal epithelial barrier. .LS174T cells are human colon adenocarcinoma cells, with the phenotype and characteristics of goblet cells, which can synthesize and secrete mucin and other secretory products associated with mucin. It can be used to study the functional.LS174T cells of goblet cells and an epithelial cell, which can be used to study the function of the intestinal epithelial barrier. Mental stress is the intestinal tract. As a risk factor for barrier destruction, it is still unclear how mental stress can regulate the function of goblet cells by CRF and the close connexin between epithelium and influence the intestinal barrier. We suspect that CRF can break through the expression of the secretory products of the intestinal goblet cells and the expression of the interepithelial tight connexin. Function of a bad intestinal barrier. Objective to investigate the effect of corticotropin releasing hormone (corticosteroid) on the intestinal barrier by regulating the secretory products of intestinal goblet cells and interepithelial tight connexin. Methods 1. LS174T cells were cultured in human colon adenocarcinoma cells, and adrenocorticotropin with different concentrations of 10-9, 10-10, 10-11, 10-12 mol/L) was used. The release hormone (CRF) acts on LS174T cells for 6 hours and PBS as the control group; 10-10 mol/L CRF acts on LS174T cells for 6 hours or 24 hours. PBS as a control group.2. uses Real-Time qPCR technique to observe the influence of CRF on the secretion of the secretory products of the LS174T cells. The best concentration of.3. using Real-Time qPCR technique to observe the effects of CRF on MUC2, FUT2, TFF3, claudin-1, Claudin-2, occludin, and the level of expression in LS174T cells. The effect of.5. on the expression of MUC2 protein in LS174T cells by immunofluorescence staining and ELISA. Results 1. Real-Time qPCR results showed that, compared with the PBS control group, the different concentrations of CRF had different effects on MUC2 in LS174T cells and 10-10 of the intestinal goblet cells. The effect of secretory products on.2. Real-Time qPCR showed that the expression of MUC2 (P0.01), FUT2 (P 0.001) and TFF3 (0.0001) decreased after 6 hours of CRF in LS174T cells. The result of mRNA expression decreased (P0.001).3. cell immunofluorescence and ELISA showed that, compared with PBS control group, the protein expression of MUC2 was reduced (P0.05) after CRF action in LS174T cells for 6 hours, but the expression of MUC2 was no significant difference after the action of CRF for 24 hours. After 6 hours of LS174T cells, the protein expression of TFF3 was decreased (P 0.001). After 24 hours of CRF, the protein levels of FUT2 and TFF3 were decreased (P0.001).5. Real-Time qPCR results showed that 10-10 mol/L CRF acted on the cells after 6 hours. After the action of CRF for 24 hours (P0.001),.6. Western blot showed that the protein expression of Claudin-2 increased (p0.001) after the action of CRF on LS174T cells for 6 hours, and the expression of occludin and ZO-1 proteins decreased after the action of CRF for 24 hours, and the protein expression and protein expression were compared with those of the control group. There is no significant difference (NS). Conclusion 1.CRF can downregulate the expression of MUC2, FUT2 and TFF3, which leads to the changes in the composition of the intestinal mucosal barrier. The adverse.2.CRF to maintain the intestinal mucosal barrier function can increase the Claudin-2, reduce the occludin and ZO-1, lead to the changes in the composition of the intercellular tight connexin, and maintain the intestinal epithelial screen. The adverse effects of.3.CRF on the secretion of the secretory products of intestinal goblet cells and the expression of interepithelial tight connexin may be the cause of the damage of the intestinal barrier function caused by mental stress.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R574

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