【摘要】：在動(dòng)物生產(chǎn)中,往往伴隨著多種腸道疾病,給畜牧業(yè)帶來(lái)了巨大的經(jīng)濟(jì)損失。腸道作為機(jī)體最大的細(xì)菌內(nèi)毒素庫(kù),擁有完整的腸道上皮屏障,對(duì)維持上皮細(xì)胞通透性、機(jī)體內(nèi)環(huán)境的穩(wěn)態(tài)有重要作用。腸上皮細(xì)胞作為對(duì)抗腸道細(xì)菌、毒素的第一道防線,時(shí)刻接觸細(xì)菌或LPS,而致病菌的侵入或者LPS可介導(dǎo)腸上皮細(xì)胞損傷,進(jìn)而引發(fā)多種疾病的發(fā)生。益生元和益生菌通過(guò)提高機(jī)體免疫力、改善動(dòng)物胃腸道的微生態(tài)環(huán)境、抑制病原菌的粘附、改善腸道組織形態(tài),成為養(yǎng)殖行業(yè)中能取代抗生素的最有前景的產(chǎn)品之一。但益生菌的研究和應(yīng)用中還存在很多瓶頸問(wèn)題,尤其是如何使益生菌添加后迅速地定植、生長(zhǎng)以及在機(jī)體內(nèi)保持豐富的濃度。這些問(wèn)題的解決才能使其穩(wěn)定地發(fā)揮作用。魔芋甘露低聚糖是一類從魔芋中提取的功能性低聚糖,具有調(diào)節(jié)免疫防御和益生元特性,促進(jìn)益生菌的生長(zhǎng)繁殖,增強(qiáng)益生菌的作用。鑒于益生菌單獨(dú)使用中存在的問(wèn)題以及魔芋甘露低聚糖良好的益生特性及發(fā)展前景,本研究利用LPS誘導(dǎo)構(gòu)建腸上皮細(xì)胞損傷模型,篩選能夠利用魔芋甘露低聚糖的益生菌,分別探討外源添加魔芋甘露低聚糖、益生菌及其與寡糖協(xié)同對(duì)腸上皮細(xì)胞損傷模型的修復(fù)作用。主要研究結(jié)果如下:1.益生菌的培養(yǎng)與鑒定對(duì)4株試驗(yàn)菌株培養(yǎng)純化后分別進(jìn)行生理生化鑒定和革蘭氏、芽孢染色,結(jié)果發(fā)現(xiàn):試驗(yàn)菌株均為革蘭氏陽(yáng)性菌,3株含有芽孢,1株不含,其生理生化特征分別與地衣芽孢桿菌、屎腸球菌、巨大芽孢桿菌和枯草芽孢桿菌相一致。提取各菌株的基因組DNA,進(jìn)行16sr RNA擴(kuò)增、測(cè)序,結(jié)果發(fā)現(xiàn):所測(cè)的菌株序列分別與上述菌株同源性最高�；谝陨辖Y(jié)果可知,培養(yǎng)的4株菌分別為地衣芽孢桿菌、屎腸球菌、巨大芽孢桿菌和枯草芽孢桿菌。2.篩選利用魔芋甘露低聚糖的益生菌菌株將3種不同濃度(1 g/L、1.5 g/L和2g/L)的魔芋甘露低聚糖與各菌株混合培養(yǎng),利用紫外分光光度計(jì)檢測(cè)魔芋甘露低聚糖對(duì)益生菌生長(zhǎng)的影響,篩選出能有效利用寡糖的益生菌菌株。研究結(jié)果發(fā)現(xiàn):與對(duì)照組相比,魔芋甘露低聚糖對(duì)枯草芽孢桿菌有顯著的促生長(zhǎng)作用,且魔芋甘露低聚糖的最佳作用濃度為2 g/L,兩者最佳反應(yīng)時(shí)間是24 h。3.腸上皮細(xì)胞損傷模型的構(gòu)建利用不同濃度的LPS刺激Caco-2細(xì)胞,q PCR檢測(cè)炎性因子IL-1β和TNF-α的表達(dá),結(jié)果發(fā)現(xiàn):與對(duì)照組相比,TNF-α和IL-1β基因的表達(dá)顯著上調(diào),且當(dāng)LPS濃度達(dá)1μg/m L,作用時(shí)間6 h時(shí),上調(diào)最為顯著。進(jìn)而利用MTT法檢測(cè)Caco-2細(xì)胞活性,結(jié)果發(fā)現(xiàn):與對(duì)照組相比,1μg/m L LPS處理組的Caco-2細(xì)胞活性顯著降低。利用實(shí)時(shí)細(xì)胞分析儀檢測(cè)Caco-2細(xì)胞阻抗的變化,LPS處理組阻抗值顯著下調(diào),這表明LPS能增加Caco-2細(xì)胞的通透性。用W estern blot檢測(cè)ZO-1在蛋白水平的表達(dá),結(jié)果顯示,ZO-1表達(dá)顯著下調(diào),表明L PS刺激能引起上皮緊密連接的損傷。綜合以上結(jié)果,表明1μg/m L LPS刺激Caco-2細(xì)胞,作用時(shí)間6 h,能成功構(gòu)建腸上皮細(xì)胞的損傷的細(xì)胞模型。4.魔芋甘露低聚糖和益生菌對(duì)腸上皮細(xì)胞損傷的修復(fù)及協(xié)同作用外源添加不同濃度的魔芋甘露寡糖、益生菌及其復(fù)合物于腸上皮損傷細(xì)胞中,MTT法檢測(cè)細(xì)胞活性,q PCR檢測(cè)IL-6,TNF-α,IL-1β,MUC-2,Claudin-1,ZO-1 m RNA表達(dá),W estern blot檢測(cè)ZO-1蛋白表達(dá)。結(jié)果發(fā)現(xiàn),與LPS損傷組相比,寡糖添加組、益生菌添加組、寡糖與菌聯(lián)合添加組的Caco-2細(xì)胞活性顯著上調(diào),TNF-α,IL-6,IL-1βm RNA表達(dá)顯著下調(diào),ZO-1,Claudin-1,MUC-2 m RNA表達(dá)顯著上調(diào),ZO-1蛋白表達(dá)上調(diào)。與益生菌添加組相比,寡糖益生菌聯(lián)合添加組的Claudin-1,ZO-1,M UC-2 m RNA表達(dá)顯著上調(diào);與寡糖添加組相比,寡糖益生菌聯(lián)合添加組Caco-2細(xì)胞活性顯著上調(diào)。表明益生菌、寡糖及兩者共同作用均能對(duì)LPS引起的Caco-2細(xì)胞損傷進(jìn)行修復(fù),且寡糖與益生菌共同作用比益生菌和寡糖單獨(dú)使用的修復(fù)作用在某些指標(biāo)上更加顯著。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S856.4

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