促肾上腺皮质激素释放激素对肠道屏障的影响研究

发布时间：2018-05-14 00:30

本文选题：促肾上腺皮质激素释放激素 + 肠粘膜屏障　；参考：《山东大学》2016年硕士论文

【摘要】：研究背景肠易激综合征(irritable bowel syndrome, IBS)是指伴有反复的腹痛和肠道习惯改变,没有可解释症状的器质性病变存在的慢性功能性消化道疾病,是研究最多的功能性胃肠病(functional gastrointestinal disorders, FGIDs)之一,其发病可能与精神应激和脑肠轴功能紊乱有关。精神应激可以通过破坏脑肠轴,导致肠道屏障的破坏和肠道通透性的增加,从而引起或者加剧肠道疾病的发生发展,比如压力可以加重IBS的症状。在应激过程中,脑肠轴的一种主要激素促肾上腺皮质激素释放激素(corticotropin releasing factor, CRF)的含量是增加的。肠道屏障包括粘液屏障和上皮屏障,对肠道功能的维持非常重要。杯状细胞合成的一些分泌产物是肠道粘膜屏障的重要组成成分。上皮间的紧密连接蛋白是肠道上皮屏障的重要组成成分。LS174T细胞是人结肠腺癌细胞,有杯状细胞的表型和特性,能够合成并分泌粘蛋白和与粘蛋白有关的其他分泌产物,可以用来研究杯状细胞的功能。LS174T细胞也是一种上皮细胞,可以用来研究肠道上皮屏障的功能。精神应激是肠道屏障破坏的一个危险因素,那么精神应激如何通过CRF调节杯状细胞的功能以及上皮间的紧密连接蛋白而影响肠道屏障的具体机制还尚不清楚,我们猜想,CRF可以通过改变肠道杯状细胞的分泌产物和上皮间紧密连接蛋白的表达从而破坏肠道屏障的功能。目的探讨促肾上腺皮质激素释放激素通过调节肠道杯状细胞的分泌产物和上皮间紧密连接蛋白对肠道屏障的影响。方法1.培养人结肠腺癌细胞LS174T细胞,应用不同浓度10-9, 10-10, 10-11, 10-12 mol/L)的促肾上腺皮质激素释放激素(CRF)作用于LS174T细胞6小时,PBS作为对照组；10-10 mol/L的CRF作用于LS174T细胞6小时或24小时,PBS作为对照组。2.利用Real-Time qPCR技术观察不同浓度的CRF对LS174T细胞分泌产物相关基因MUC2, FUT2和TFF3的mRNA水平表达量的影响,以确定CRF作用的最佳浓度。3.利用Real-Time qPCR技术观察10-10 mol/L的CRF对LS174T细胞中MUC2, FUT2, TFF3, claudin-1, claudin-2, occludin和ZO-1的mRNA水平表达量的影响。4.利用Western blot技术观察10-10 mol/L的CRF对LS174T细胞中FUT2,TFF3, claudin-1, claudin-2, occludin和ZO-1的蛋白水平表达量的影响。5.利用免疫荧光染色和ELISA技术观察CRF对LS174T细胞中MUC2蛋白水平表达量的影响。结果1. Real-Time qPCR结果显示,与PBS对照组相比,不同浓度的CRF对LS174T细胞中MUC2, FUT2口TFF3有不同的影响,10-10 mol·L-1的CRF对肠道杯状细胞分泌产物的影响相对明显。2. Real-Time qPCR结果显示,10-10 mol·L-1的CRF作用于LS174T细胞6小时后,MUC2 (P0.01), FUT2 (P 0.001)和TFF3 (P 0.0001)的mRNA表达量均下降；CRF作用于LS174T细胞24小时后,与对照组相比,MUC2和FUT2的mRNA表达量无明显差异(NS),而TFF3的mRNA表达量仍下降(P0.001)。3.细胞免疫荧光和ELISA的结果显示,与PBS对照组相比,CRF作用于LS174T细胞6小时后,MUC2的蛋白表达量降低(P0.05),而CRF作用于细胞24小时后,MUC2的表达无显著性差异(NS)。4. Western blot结果显示,与PBS对照组相比,CRF作用LS174T细胞6小时后,TFF3的蛋白表达量是下降的(P 0.001), CRF作用24小时后,FUT2和TFF3的蛋白水平均是下降的(P0.001)。5. Real-Time qPCR结果显示,10-10 mol/L CRF作用于LS174T细胞6小时后,claudin-2的mRNA表达水平增加(p0.001),而occludin和ZO-1的mRNA表达水平在CRF作用24小时后下降(P0.001)。6. Western blot结果显示,CRF作用于LS174T细胞6小时后,claudin-2的蛋白表达量增加(p0.001),而CRF作用24小时后,occludin和ZO-1的蛋白表达量下降(P0.001)。7.CRF作用于LS174T细胞6小时后,claudin-1的mRNA和蛋白表达量与PBS对照组相比均无显著性差异(NS)。结论1.CRF可以下调MUC2, FUT2和TFF3的表达量,导致肠粘膜屏障的组成成分发生改变,对维持肠粘膜屏障功能不利。2.CRF可以上调claudin-2,下调occludin和ZO-1,导致细胞间紧密连接蛋白的成分发生改变,对维持肠道上皮屏障的完整性不利。3.CRF对肠道杯状细胞分泌产物的分泌以及上皮间紧密连接蛋白的表达的影响可能是精神应激导致肠道屏障功能破坏的原因。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R574

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