三种动物回肠总菌及乳酸菌对家兔隐窝和小鼠DCs炎性反应的影响

发布时间：2018-05-11 06:31

本文选题：隐窝细胞 + DC　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：家兔肠道易受感染,进而导致严重的炎症和随后高的死亡率。肠道中的细菌包括各种共生菌代谢产物和菌体成分可以诱导肠道炎症反应。研究证实革兰氏阴性菌菌体比阳性菌菌体更容易引起炎症。与猪和鸡等单胃动物不同,采食后的家兔各肠段的主要菌门是革兰氏阴性菌拟杆菌,且革兰氏阳性菌乳酸菌在总菌中所占比例很低。本论文比较健康成年猪、鸡和家兔回肠总菌对培养的家兔隐窝细胞及DC炎症反应的诱导作用,并研究本实验室已经筛选的几株兔源乳酸菌对肠道上皮中隐窝细胞和树突状细胞的炎性调节作用,以探讨家兔特有的肠道菌群是否会导致其肠道更易发生炎症,以及乳酸菌是否可缓解炎性反应,且不同乳酸菌的缓解作用是否存在差异,两种不同细胞类型对抗原的炎性反应的差异,为家兔筛选益生菌,提高仔兔免疫力和减少仔兔肠炎发生提供理论依据。主要的研究内容及结果如下:1.试验一从未饲喂抗菌素的健康6月龄关中黑猪、42日龄科宝肉鸡和60日龄成年獭兔回肠中收集内容物,每种动物3个个体的回肠内容物等量混样提取菌群基因组DNA,Illumina HiSeq测序对比分析猪、鸡和家兔回肠总菌组成。结果显示,家兔回肠菌群第一优势菌门是拟杆菌门,占总菌群的百分比为89.32%;猪和鸡回肠菌群最优菌门是厚壁菌门,分别占80.79%和88.50%。三种动物回肠内容物中乳杆菌属丰度分别为19.19%、81.19%和0.72%。Beta多样性指数显示猪和鸡回肠菌群组成接近,家兔与两者差异较大。2.试验二(1)对试验一中猪、鸡及家兔回肠总菌灭活后作用于培养的家兔隐窝细胞,空白对照组添加PBS,热灭活总菌处理隐窝6 h后收集细胞,测定相关基因的表达。结果如下:与对照组相比,猪和鸡回肠灭活总菌显著提高家兔隐窝细胞的TLR2、TLR3和TLR4的表达(P0.05);家兔回肠总菌能显著提高TLR4的表达(P0.01)。各处理组对TLR9均无显著影响(P0.05)。三种总菌均显著提高了促炎性因子TNF-α和IL-6的表达(P0.05)。鸡回肠总菌对抗炎性因子IL-10的表达刺激作用比其他组显著(P0.05),且猪和鸡回肠总菌提高了IFN-β的表达(P0.05),猪回肠总菌比家兔回肠总菌有增加IL-10表达的趋势(P0.1),但家兔回肠总菌对抗炎因子IL-10的表达没有促进作用(P0.05),甚至有降低IFN-β表达的趋势(P0.1)。(2)用实验室前期分离的兔源植物乳杆菌L.p2、干酪乳杆菌L.a1和屎肠球菌E.f2,以及购买的大肠杆菌E.coli K88灭活后对仔兔隐窝细胞进行刺激6 h后收集细胞,测定相关基因的表达。结果如下:三株乳酸菌显著提高了TLR2、TLR3和IFN-β的表达(P0.05)。大肠杆菌E.coli K88显著提高了TLR4表达(P0.05)。植物乳酸菌L.p2、屎肠球菌E.f2和大肠杆菌E.coli K88组均显著提高隐窝细胞TNF-α的表达(P0.05)。各处理均显著提高了IL-6的表达(P0.05)。植物乳杆菌L.p2和屎肠球菌E.f2显著提高了IL-10的表达(P0.05),E.coli K88组IL10的表达显著低于对照组(P0.05)。三株乳酸菌中植物乳杆菌对促炎性因子的刺激作用更强。3.试验三用猪、鸡和家兔回肠总菌及兔源乳酸菌的灭活菌刺激小鼠骨髓源树突状细胞系DC2.4细胞,结果表明:(1)DC2.4细胞受到灭活L.p2刺激后,TLR2、TLR4、TNF-α和IL-10表达随着刺激时间增加呈现先升高后降低的规律,在2 h前后表达量较高。(2)猪、鸡和家兔回肠总菌刺激DC2.4细胞2 h后,猪和鸡回肠菌比家兔回肠菌显著提高TLR3表达(P0.05)。猪和鸡回肠菌显著提高了TLR2和TLR4的表达(P0.05),家兔回肠菌有提高TLR2的趋势(P0.1),但对TLR3无显著影响(P0.05)。各处理组对TLR9表达无显著影响(P0.05),均显著提高了TNF-α及IL-10的表达(P0.05),但家兔回肠总菌对TNF-α表达刺激作用显著高于猪和鸡回肠总菌。鸡回肠总菌显著提高了IL-6和IFN-β的表达(P0.05),猪回肠总菌有提高IL-6和IFN-β基因表达的趋势(P0.1),但是家兔回肠总菌对IFN-β的表达无明显的影响(P0.05)和有提高IL-6基因表达的趋势(P0.1)。(3)在乳酸菌刺激DC2.4细胞2 h的试验中,与对照组相比,L.p2、L.a1和E.f2显著提高TLR2的表达(P0.05)。L.a1和E.f2显著提高了TLR3的表达(P0.05)。E.coli K88显著提高了TLR4和TNF-α的表达(P0.05)。L.p2和E.f2显著提高了TNF-α基因的表达(P0.05)。相比对照组,L.a1和E.f2有增加IFN-β的趋势(P0.1)。E.f2和E.coli K88显著提高了IL-10基因表达(P0.05)。综上所述,家兔回肠菌群第一优势菌门是拟杆菌门,猪和鸡第一优势菌门为厚壁菌门,且家兔回肠乳酸菌远远低于猪和鸡;与猪和鸡回肠总菌相比,家兔回肠总菌更容易使隐窝和DC2.4细胞处于炎症状态;灭活乳酸菌能够引起细胞免疫反应中抗炎性和促炎性因子的同步表达,有利于维持机体的稳态;综合促炎因子和抗炎因子表达水平,干酪乳杆菌和屎肠球菌比植物乳杆菌更有利于维持隐窝的炎性稳态,但对DC2.4细胞的刺激试验没有获得相同的结论。
[Abstract]:The intestinal tract of the rabbit is susceptible to infection, which leads to severe inflammation and subsequent high mortality. The bacteria in the intestinal tract, including the metabolites of the symbiotic bacteria and the body components, can induce intestinal inflammation. The study confirms that the Gram-negative bacteria are more prone to inflammation than the positive bacteria. The main bacteria in each segment of the rabbit were gram-negative bacilli, and the proportion of gram-positive Lactobacillus in the total bacteria was very low. In this paper, we compared the induction of the cultured rabbit's recess cells and the DC inflammatory reaction in the healthy adult pig, chicken and rabbit, and studied several strains of lactic acid bacteria that had been screened in our laboratory. The inflammatory regulation of recess cells and dendritic cells in the intestinal epithelium can be used to investigate whether the unique intestinal flora of the rabbit can cause inflammation in the intestinal tract more easily, and whether the lactic acid bacteria can alleviate the inflammatory reaction, and whether there are differences in the remission of different lactic acid bacteria and the difference of the inflammatory response of two different cell types to the antigen. It provides a theoretical basis for the screening of probiotics in rabbits, improving immunity and reducing the occurrence of enteritis in rabbits. The main contents and results are as follows: 1. the contents of black pigs in the healthy 6 month old Guanzhong, 42 day old Cobo broilers and 60 day old Rex rabbits were collected, and the contents of the ileum in 3 individual animals of each animal were collected. DNA and Illumina HiSeq sequencing were used to analyze the composition of the total bacteria in the ileum of pigs, chickens and rabbits. The results showed that the first dominant bacteria gate of the ileum was 89.32%, the percentage of the total flora was 89.32%, and the best phylum of the pig and chicken ileum was 80.79% and 88.50%. in the ileum, respectively. The abundance of lactobacilli was 19.19%, 81.19% and 0.72%.Beta diversity index showed that the composition of the pig and the chicken ileum group was close, and the difference between the rabbit and the two was two (1), which was two (1) of the experiment 1 pig, chicken and rabbit ileum inactivated by the cultured rabbit recess cells, the blank control group added PBS, the thermal inactivated total bacteria treated hidden bacteria. After 6 h, the cells were collected to determine the expression of related genes. The results were as follows: compared with the control group, the total bacteria of the pig and chicken ileum inactivated significantly increased the expression of TLR2, TLR3 and TLR4 in the recess cells of the rabbit (P0.05), and the total intestinal bacteria in the rabbit could significantly increase the expression of TLR4 (P0.01). There was no significant effect on TLR9 in each group (P0.05). The three kinds of total bacteria were significantly raised. The expression of pro-inflammatory factor TNF- A and IL-6 (P0.05). The expression of total bacteria in the ileum of chicken was significantly higher than that of the other groups (P0.05), and the expression of IFN- beta in pig and chicken ileum increased (P0.05), and the total intestinal bacteria in pig ileum had a tendency to increase the expression of IL-10 (P0.1) than that of the total ileum in rabbits (P0.1), but the total intestinal bacteria in the rabbits were against the cause of inflammation. The expression of subIL-10 was not promoted (P0.05), and even decreased the trend of IFN- beta expression (P0.1). (2) the cells were collected after 6 h stimulation of Lactobacillus plantarum L.p2, Lactobacillus casei L.a1 and Enterococcus faecium E.f2, and the purchased Escherichia coli E.coli K88 inactivated in the early laboratory, and the related genes were detected. The results were as follows: three strains of lactic acid bacteria significantly increased the expression of TLR2, TLR3 and IFN- beta (P0.05). E.coli K88 in Escherichia coli significantly increased the expression of TLR4 (P0.05). The expression of the plant Lactococcus bacteria L.p2, the E.f2 of Enterococcus faecium and the E.coli K88 group of Escherichia coli were significantly increased. 0.05). Lactobacillus plantarum L.p2 and Enterococcus faecium E.f2 significantly increased the expression of IL-10 (P0.05), and the expression of IL10 in E.coli K88 group was significantly lower than that of the control group (P0.05). The stimulation of Lactobacillus plantarum in three strains of Lactobacillus plantarum stronger.3. test three pigs, chicken and rabbit ileum total bacteria and rabbit source of lactic acid bacteria to stimulate mouse bone marrow The result of the source dendritic cell line DC2.4 cells showed that (1) the expression of TLR2, TLR4, TNF- alpha and IL-10 expression increased first and then decreased with the increase of stimulation time. (2) the expression of TLR2, TLR4, TNF- alpha and IL-10 was higher before and after the 2 h. (2) pigs, chickens and rabbit ileum bacteria stimulated DC2.4 cell 2 h, pigs and chicken ileum were more significant than those of rabbit ileum bacteria The expression of TLR3 (P0.05) was enhanced. The expression of TLR2 and TLR4 was significantly increased in pigs and chicken ileum (P0.05), and in rabbit ileum had a tendency to increase TLR2 (P0.1), but there was no significant effect on TLR3 (P0.05). The expression of TNF- alpha and the expression of TLR9 had no significant effect on the expression of TLR9 (P0.05). The expression of IL-6 and IFN- beta was significantly increased in chicken ileum (P0.05), and the trend of IL-6 and IFN- beta gene expression increased (P0.1) in pig ileum total bacteria (P0.1), but there was no obvious effect on the expression of IFN- beta (P0.05) and the trend of improving the expression of IL-6 gene (P0.1) in the total intestinal bacteria of the rabbit (3) in Lactobacillus spines. Compared with the control group, L.p2, L.a1 and E.f2 significantly increased the expression of TLR2 (P0.05).L.a1 and E.f2 significantly increased the expression of TLR3 (P0.05), compared with the control group, and the expression of TLR3 (P0.05) was significantly increased by L.p2, L.a1 and E.f2. The trend (P0.1).E.f2 and E.coli K88 significantly increased the IL-10 gene expression (P0.05). To sum up, the first dominant bacteria gate of the rabbit ileum was the pseudo bacilli gate, the first dominant bacteria gate of the pig and chicken was the thick wall bacteria gate, and the rabbit ileum lactobacillus was far lower than the pig and chicken; compared with the total bacteria of the pig and chicken ileum, the rabbit ileum was more likely to make the recess and DC. The 2.4 cells are in the inflammatory state; inactivating lactic acid bacteria can cause the synchronous expression of anti-inflammatory and proinflammatory factors in the cellular immune response, which is beneficial to maintain the homeostasis of the body; the expression level of pro-inflammatory and anti-inflammatory factors, Lactobacillus casei and Enterococcus faecium are more favorable for maintaining the inflammatory homeostasis of the recess than the Lactobacillus plantarum, but the DC2.4 is fine. The stimulation test of the cell did not obtain the same conclusion.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.291

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