魔芋甘露低聚糖与枯草芽孢杆菌对肠上皮细胞损伤的协同修复作用

发布时间：2018-09-18 20:12

【摘要】：在动物生产中,往往伴随着多种肠道疾病,给畜牧业带来了巨大的经济损失。肠道作为机体最大的细菌内毒素库,拥有完整的肠道上皮屏障,对维持上皮细胞通透性、机体内环境的稳态有重要作用。肠上皮细胞作为对抗肠道细菌、毒素的第一道防线,时刻接触细菌或LPS,而致病菌的侵入或者LPS可介导肠上皮细胞损伤,进而引发多种疾病的发生。益生元和益生菌通过提高机体免疫力、改善动物胃肠道的微生态环境、抑制病原菌的粘附、改善肠道组织形态,成为养殖行业中能取代抗生素的最有前景的产品之一。但益生菌的研究和应用中还存在很多瓶颈问题,尤其是如何使益生菌添加后迅速地定植、生长以及在机体内保持丰富的浓度。这些问题的解决才能使其稳定地发挥作用。魔芋甘露低聚糖是一类从魔芋中提取的功能性低聚糖,具有调节免疫防御和益生元特性,促进益生菌的生长繁殖,增强益生菌的作用。鉴于益生菌单独使用中存在的问题以及魔芋甘露低聚糖良好的益生特性及发展前景,本研究利用LPS诱导构建肠上皮细胞损伤模型,筛选能够利用魔芋甘露低聚糖的益生菌,分别探讨外源添加魔芋甘露低聚糖、益生菌及其与寡糖协同对肠上皮细胞损伤模型的修复作用。主要研究结果如下:1.益生菌的培养与鉴定对4株试验菌株培养纯化后分别进行生理生化鉴定和革兰氏、芽孢染色,结果发现:试验菌株均为革兰氏阳性菌,3株含有芽孢,1株不含,其生理生化特征分别与地衣芽孢杆菌、屎肠球菌、巨大芽孢杆菌和枯草芽孢杆菌相一致。提取各菌株的基因组DNA,进行16sr RNA扩增、测序,结果发现:所测的菌株序列分别与上述菌株同源性最高。基于以上结果可知,培养的4株菌分别为地衣芽孢杆菌、屎肠球菌、巨大芽孢杆菌和枯草芽孢杆菌。2.筛选利用魔芋甘露低聚糖的益生菌菌株将3种不同浓度(1 g/L、1.5 g/L和2g/L)的魔芋甘露低聚糖与各菌株混合培养,利用紫外分光光度计检测魔芋甘露低聚糖对益生菌生长的影响,筛选出能有效利用寡糖的益生菌菌株。研究结果发现:与对照组相比,魔芋甘露低聚糖对枯草芽孢杆菌有显著的促生长作用,且魔芋甘露低聚糖的最佳作用浓度为2 g/L,两者最佳反应时间是24 h。3.肠上皮细胞损伤模型的构建利用不同浓度的LPS刺激Caco-2细胞,q PCR检测炎性因子IL-1β和TNF-α的表达,结果发现:与对照组相比,TNF-α和IL-1β基因的表达显著上调,且当LPS浓度达1μg/m L,作用时间6 h时,上调最为显著。进而利用MTT法检测Caco-2细胞活性,结果发现:与对照组相比,1μg/m L LPS处理组的Caco-2细胞活性显著降低。利用实时细胞分析仪检测Caco-2细胞阻抗的变化,LPS处理组阻抗值显著下调,这表明LPS能增加Caco-2细胞的通透性。用W estern blot检测ZO-1在蛋白水平的表达,结果显示,ZO-1表达显著下调,表明L PS刺激能引起上皮紧密连接的损伤。综合以上结果,表明1μg/m L LPS刺激Caco-2细胞,作用时间6 h,能成功构建肠上皮细胞的损伤的细胞模型。4.魔芋甘露低聚糖和益生菌对肠上皮细胞损伤的修复及协同作用外源添加不同浓度的魔芋甘露寡糖、益生菌及其复合物于肠上皮损伤细胞中,MTT法检测细胞活性,q PCR检测IL-6,TNF-α,IL-1β,MUC-2,Claudin-1,ZO-1 m RNA表达,W estern blot检测ZO-1蛋白表达。结果发现,与LPS损伤组相比,寡糖添加组、益生菌添加组、寡糖与菌联合添加组的Caco-2细胞活性显著上调,TNF-α,IL-6,IL-1βm RNA表达显著下调,ZO-1,Claudin-1,MUC-2 m RNA表达显著上调,ZO-1蛋白表达上调。与益生菌添加组相比,寡糖益生菌联合添加组的Claudin-1,ZO-1,M UC-2 m RNA表达显著上调;与寡糖添加组相比,寡糖益生菌联合添加组Caco-2细胞活性显著上调。表明益生菌、寡糖及两者共同作用均能对LPS引起的Caco-2细胞损伤进行修复,且寡糖与益生菌共同作用比益生菌和寡糖单独使用的修复作用在某些指标上更加显著。
[Abstract]:In animal production, intestinal diseases often accompany with a variety of intestinal diseases, which bring enormous economic losses to animal husbandry. As the largest bacterial endotoxin pool in the body, intestinal tract has a complete intestinal epithelial barrier, which plays an important role in maintaining the permeability of epithelial cells and the homeostasis of the body's internal environment. Probiotics and probiotics can improve the microenvironment of animal gastrointestinal tract, inhibit the adhesion of pathogenic bacteria, improve the intestinal tissue morphology, and become the best choice in the aquaculture industry. Among the most promising alternatives to antibiotics, however, there are still many bottlenecks in the research and application of probiotics, especially how to rapidly colonize, grow and maintain abundant concentrations in the body after the addition of probiotics. The extracted functional oligosaccharides can regulate immune defense and prebiotic properties, promote the growth and reproduction of probiotics, and enhance the role of probiotics. Probiotics which can utilize konjac mannan oligosaccharides were selected to study the effects of exogenous konjac mannan oligosaccharides, probiotics and their cooperation with oligosaccharides on the repair of intestinal epithelial cell injury model. The main results are as follows: 1. The culture and identification of probiotics were carried out on the physiological and biochemical identification of the four strains after culture and purification, respectively, and Gram. Bacillus subtilis, Enterococcus faecium, Bacillus megalobacter and Bacillus subtilis were detected. The genomic DNA of all the strains was extracted, amplified and sequenced by 16sr RNA. The results showed that the sequence of the tested strains was identical with that of Bacillus licheniformis, Enterococcus faecium, Bacillus megalobacter and Bacillus subtilis. The four strains were Bacillus licheniformis, Enterococcus faecium, Bacillus megaterium and Bacillus subtilis. 2. Three different concentrations of konjac mannan oligosaccharides (1 g/L, 1.5 g/L and 2 g/L) were mixed with each strain. The effects of konjac mannan oligosaccharides on the growth of probiotics were detected by ultraviolet spectrophotometer. The results showed that konjac mannan oligosaccharides could promote the growth of Bacillus subtilis significantly, and the optimum concentration of konjac mannan oligosaccharides was 2 g/L. The optimum reaction time was 24 h.3. The model of intestinal epithelial cell injury was constructed. Different concentrations of LPS were used to stimulate Caco-2 cells. The expression of inflammatory factors IL-1beta and TNF-alpha was detected by q-PCR. The results showed that the expression of TNF-alpha and IL-1beta genes were significantly up-regulated compared with the control group, and the up-regulated was most obvious when the concentration of LPS reached 1 ug/ml and the duration of action was 6 h. Furthermore, the activity of Caco-2 cells was detected by MTT assay. The results showed that compared with the control group, the activity of Caco-2 cells was significantly decreased in the treatment group treated with 1 ug/m LPS. The impedance of Caco-2 cells was significantly decreased by real-time cell analyzer, which indicated that LPS could increase the permeability of Caco-2 cells. The results showed that the expression of ZO-1 was down-regulated at the protein level, suggesting that LPS stimulation could induce the injury of tight junction of epithelium. The expression of IL-6, TNF-a, IL-1beta, MUC-2, Claudin-1, ZO-1 m RNA and ZO-1 protein were detected by q-PCR, and the expression of ZO-1 protein was detected by W estern blot. The expression of TNF-a, IL-6, IL-1 beta m RNA, ZO-1, Claudin-1, MUC-2 m RNA and ZO-1 protein were significantly up-regulated in the probiotic group and the oligosaccharide-plus group. Compared with the probiotic group, the expression of Claudin-1, ZO-1 and MUC-2 m RNA was significantly up-regulated in the oligosaccharide-plus group. Compared with the oligosaccharide supplementation group, the activity of Caco-2 cells in the probiotics-oligosaccharide combination group was significantly increased, suggesting that probiotics, oligosaccharides and their combined effects could repair LPS-induced damage to Caco-2 cells, and the combined effects of oligosaccharides and probiotics were more significant than probiotics and oligosaccharides alone in some indicators.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S856.4

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