嗜酸乳杆菌及长双歧杆菌上清对肠上皮细胞5-羟色胺转运体表达调控研究

发布时间：2018-05-25 13:45

本文选题：嗜酸乳杆菌上清 + 长双歧杆菌上清　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:肠易激综合征(Irritable Bowel Syndrome,IBS)是一种以腹痛、腹胀、腹部不适为特征症状,常伴有排便频率和(或)粪便性状改变的临床常见消化道功能性疾病。近年研究表明,5-羟色胺(5-hydroxytryp-tamine,5-HT)及其转运体(Serotonin Transporter,SERT)的代谢紊乱可能导致内脏感觉异常、肠道运动紊乱,在IBS的发病中有重要作用。目前研究表明SERT表达的高低除受SERT基因多态性的影响外,还受肠道微生态的影响。大量临床试验证明益生菌可以改善IBS患者腹痛、腹胀等症状,对于治疗肠易激综合症疗效显著。乳酸杆菌和双歧杆菌是IBS中最常研究的益生菌,其中嗜酸乳杆菌及长双歧杆菌已经作为益生菌制剂应用于临床。然而益生菌制剂改善IBS症状的具体作用机制尚不明确。我们之前的研究表明鼠李糖乳杆菌上清可以上调肠道上皮细胞和小鼠结肠组织SERT的表达,然而目前尚无研究证实嗜酸乳杆菌和长双歧杆菌上清是否也能够调节SERT的表达。本研究的目的是观察嗜酸乳杆菌和长双歧杆菌上清对肠道上皮细胞SERT mRNA及SERT蛋白的影响,从而为研究益生菌治疗IBS作用机制提供新的理论依据。方法:本实验分别用1:100、1:50、1:20 3种不同稀释浓度的嗜酸乳杆菌(ATCC4356)上清和长双歧杆菌(ATCC 15707)上清刺激HT-29和Caco-2细胞12小时和24小时,然后应用RT-PCR检测细胞的SERT mRNA的表达水平变化,应用Western-Blot检测细胞的SERT蛋白的表达水平变化。统计学分析应用SPSS 17.0统计软件:用均数±标准差(±s)来表示计量资料。应用单因素方差分析来比较3组以上计量资料的组间差异,用LSD方法检验方差齐性数据,用Dunnett方法检验方差不齐数据。以P0.05表示差异有统计学意义。结果:1:100,1:50和1:20稀释的嗜酸乳杆菌上清刺激HT-29细胞12小时和24小时,刺激组SERT mRNA表达水平分别是对照组的1.80,2.24,2.28,和2.04,2.30,2.80倍(P0.05,P0.05,P0.05,和P0.05,P0.05,P0.05);1:100,1:50和1:20稀释的嗜酸乳杆菌上清刺激Caco-2细胞12小时和24小时,刺激组SERT mRNA表达水平分别是对照组的1.20,1.22,1.61,和1.16,1.50,2.13倍(P0.05,P0.05,P0.05,和P0.05,P0.05,P0.05);1:100,1:50和1:20稀释的长双歧杆菌上清刺激HT-29细胞12小时和24小时,刺激组SERT mRNA表达水平分别是对照组的0.96,1.90,2.50,和2.58,2.19,3.21倍(P0.05,P0.05,P0.05,和P0.05,P0.05,P0.05);1:100,1:50和1:20稀释的长双歧杆菌上清刺激Caco-2细胞12小时和24小时,刺激组SERT mRNA表达水平分别是对照组的1.25,2.06,1.83和1.84,2.23,2.13倍(P0.05,P0.05,P0.05,和P0.05,P0.05,P0.05)。各浓度嗜酸乳杆菌与长双歧杆菌上清均上调HT-29细胞和Caco-2细胞SERT蛋白的表达,上调水平与相应的mRNA上调水平大致相同。结论:(1)嗜酸乳杆菌及长双歧杆菌上清可以上调肠道上皮细胞SERTmRNA及SERT蛋白的表达;(2)嗜酸乳杆菌及长双歧杆菌上清上调SERTm RNA和SERT蛋白的表达具有时间依赖性和浓度依赖性。
[Abstract]:Objective: Irritable Bowel Syndrome syndrome (IBS) is a common clinical gastrointestinal functional disease characterized by abdominal pain, abdominal distension and abdominal discomfort, often accompanied by defecation frequency and / or fecal changes. Recent studies have shown that the metabolic disorder of serotonin transporter serotonin (serotonin) and serotonin transporter (serotonin) may lead to visceral sensory disorder and intestinal motility disorder, which may play an important role in the pathogenesis of IBS. Current studies have shown that the expression of SERT is affected not only by the polymorphism of SERT gene, but also by intestinal microecology. A large number of clinical trials have proved that probiotics can improve abdominal pain, abdominal distension and other symptoms in patients with IBS, and it is effective in the treatment of irritable bowel syndrome. Lactobacillus and Bifidobacterium are the most commonly studied probiotics in IBS. Lactobacillus acidophilus and Bifidobacterium long have been used as probiotics in clinic. However, the mechanism of probiotics in improving IBS symptoms is unclear. Our previous studies have shown that the supernatants of Lactobacillus rhamnoides can up-regulate the expression of SERT in intestinal epithelial cells and colonic tissues of mice. However, there is no study on whether the supernatants of Lactobacillus acidophilus and Bifidobacterium can also regulate the expression of SERT. The purpose of this study was to observe the effects of supernatants of Lactobacillus acidophilus and Bifidobacterium longidus on SERT mRNA and SERT protein in intestinal epithelial cells, and to provide a new theoretical basis for studying the mechanism of probiotics in treating IBS. Methods: in this experiment, HT-29 and Caco-2 cells were stimulated with 1: 100 and 1: 50: 20 supernatants of 1: 100, 1: 50, 1: 20 with different diluted concentrations of Lactobacillus acidophilus (ATCC4356) and Bifidobacterium longidus (ATCC15707) for 12 and 24 hours, respectively. The expression of SERT mRNA was detected by RT-PCR. The expression of SERT protein was detected by Western-Blot. Statistical analysis using SPSS 17.0 statistical software: mean 卤standard deviation (卤s) to represent the measurement data. Univariate analysis of variance was used to compare the differences among three groups of measurement data. LSD method was used to test the homogeneity data of variance and Dunnett method was used to test the uneven data. The difference was statistically significant with P0.05. Results HT-29 cells were stimulated by the supernatant of Lactobacillus acidophilus diluted at 1: 1: 100 and 1:20 for 12 hours and 24 hours, respectively. The SERT mRNA expression levels in the stimulated group were 1.80 / 2.242.28 and 2.042.302.80 times as compared with those in the control group, respectively, and the Caco-2 cells were stimulated by the supernatants of P0.05P0.05P0.05P0.05P0.05P0.05: 50 and 1:20 diluted by Lactobacillus acidophilus for 12 hours and 24 hours, respectively, in which the supernatant of Lactobacillus acidophilus diluted at 1: 1: 50 and 1:20 stimulated Caco-2 cells for 12 hours and 24 hours, respectively. The expression levels of SERT mRNA in the stimulated group were 1.20 ~ 1.22 / 1.61, 1.16 ~ 1.50 ~ 1.50 ~ 2.13 times, P _ (0.05) P _ (0.05) P _ (0.05) and P _ (0.05) P _ (0.05) P ~ (0.05) respectively, and the supernatant of 1: 100: 1: 50 and 1:20 diluted bifidobacterium longus stimulated HT-29 cells for 12 and 24 hours, respectively. The expression levels of SERT mRNA in the stimulated group were 0.96 ~ 1.90 ~ 2.50, 2.58 ~ 2.19 and 3.21 times of the control group, respectively, and the levels of SERT mRNA expression in the stimulated group were as follows: P0.05, P0.05, P0.05, and P0.05P0.05, respectively, and the SERT mRNA expression levels in the stimulated group were 12 hours and 24 hours after the supernatant of P0.05: 100: 50 and 1:20 diluted by Bifidobacterium longii, respectively. The SERT mRNA expression levels in the stimulated group were 1.252.061.83 and 1.82.232.13 times of those in the control group, respectively. The levels of SERT mRNA expression in the stimulated group were 1.252.06.83 and 1.82.232.13 times that of the control group, respectively, and the expression levels of SERT mRNA in the stimulated group were 1.252.06.83 and 1.82.232.13 times as much as that in the control group. The supernatants of Lactobacillus acidophilus and Bifidobacterium longidus upregulated the expression of SERT protein in HT-29 and Caco-2 cells, and the up-regulation level was approximately the same as that of mRNA. Conclusion the supernatants of Lactobacillus acidophilus and Bifidobacterium longifera can up-regulate the expression of SERTmRNA and SERT protein in intestinal epithelial cells. The supernatants of Lactobacillus acidophilus and Bifidobacterium can up-regulate the expression of SERTm RNA and SERT proteins in a time-and concentration-dependent manner.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574.4

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