卵黄抗体对鼠伤寒沙门氏菌感染小鼠肠道免疫应答的调节作用
发布时间：2019-06-18 15:18
【摘要】：当动物机体受到外来病原菌入侵时,免疫系统被激活以抵御病原菌对机体的伤害。然而免疫系统的高度活化会导致动物采食量及体重下降等免疫应激现象,这给养殖业造成巨大经济损失。因此如何缓解免疫应激具有非常重要的实际意义。抗生素可有效改善动物的生长状况,但随着动物产品中药物残留和细菌耐药性等问题的出现,迫切需要寻找一种既能有效控制养殖动物疾病,又不带来食品安全问题和环境隐患的新产品以替代抗生素。研究表明,卵黄抗体可用于动物感染性疾病的防治,因其具有安全、高效、特异性强等优点,所以被认为是最有前景的新一代绿色、安全型抗生素替代品。然而,关于IgY的作用机理现在还未阐明,IgY在抑制病原菌的同时,能否对动物机体的免疫系统起到调节作用,有待于进一步的研究。本课题在前期研究的基础上,通过体内实验分析IgY对鼠伤寒沙门氏菌攻毒小鼠肠道黏膜各种T淋巴细胞的影响。此外,通过体外实验还考察了IgY对巨噬细胞吞噬活性和树突细胞成熟度的影响。IgY对攻毒小鼠肠道黏膜各种T淋巴细胞的影响：昆明小鼠随机分为4组,用链霉素预处理2d,12h后灌胃PBS、非特异性IgY和特异性IgY,持续3 d,在此期间观察小鼠的状态、死亡率和体重。3d后解剖小鼠并取小肠,分离肠道派尔淋巴结、上皮层以及固有层的淋巴细胞,用流式细胞仪检测各种淋巴细胞的数量变化。结果表明,鼠伤寒沙门氏菌攻毒后,小鼠精神状态萎靡,死亡率达到60%,体重明显减轻,派尔淋巴结出现肿大,CD3+T细胞和CD8+T细胞分别提高10%和14%。上皮内淋巴细胞出现大量增加,CD8+T细胞和TCRγδ+T细胞分别增加18%和16%。固有层中CD4+T细胞和CD8+T细胞升高8%和12%。口服特异性IgY后,小鼠精神状态好转,死亡率降至20%,但体重无明显变化。派尔淋巴结肿大状况好转,并且派尔淋巴结中总的T细胞(CD3+T细胞)降低6.5%,基本恢复到正常水平。特异性IgY还可显著降低上皮层中CD8+T细胞和TCRγδ+T细胞的比例,分别降低12%和10%。固有层中的淋巴细胞在特异性IgY的作用下,也显著降低,其中CD4+T细胞和CD8+T细胞分别减少7%和12%。这表明,特异性IgY可缓解鼠伤寒沙门氏菌攻毒引起的炎症反应,对机体起到保护作用。IgY对巨噬细胞吞噬活性的影响：用FITC标记鼠伤寒沙门氏菌,然后巨噬细胞与细菌共培养3h后,检测巨噬细胞的吞噬率及NO的释放量。结果表明,非特异性IgY和特异性IgY可显著提高巨噬细胞对细菌的吞噬率,分别提高20%和33%。并且特异性IgY显著减少了巨噬细胞NO的分泌,使NO的分泌量由原先的26μmol/L降低至14 μmol/L.这表明IgY可增强巨噬细胞对细菌的吞噬率并减少炎症因子的分泌。IgY对树突细胞成熟度的影响：从小鼠骨髓中分离原代细胞,用10 ng/mL rGM-CSF和rIL-4诱导分化成树突细胞,收集第7d的未成熟树突细胞。将鼠伤寒沙门氏菌与未成熟树突细胞以及特异性IgY共同培养3 h,用流式细胞仪检测树突细胞表面标志的变化。结果表明,鼠伤寒沙门氏菌攻毒后树突细胞表面标志MHCⅡ, CD40, CD80, CD83和CD86分别降低11%、3%、4%、5%和5%,而加入特异性IgY后分别升高20%、9%、11%、15%和5%。这说明攻毒后,鼠伤寒沙门氏菌引起树突细胞的凋亡,而加入特异性IgY后,可以减少细胞的凋亡并促进树突细胞的成熟。
[Abstract]:When the animal body is invaded by foreign pathogenic bacteria, the immune system is activated to resist the harm of the pathogenic bacteria to the body. However, the high activation of the immune system can lead to the immune stress phenomena such as the feed intake and the weight loss of the animals, which causes great economic losses to the breeding industry. So how to alleviate the immune stress has very important practical significance. The antibiotic can effectively improve the growth condition of the animal, but with the problems of drug residue and bacterial drug resistance in the animal product, the invention urgently needs to find a new product which not only can effectively control the disease of the culture animals, but also does not bring food safety problems and environmental hidden dangers to replace the antibiotics. The research shows that the yolk antibody can be used for preventing and treating infectious diseases of animals, and is considered to be the most promising new generation of green and safe antibiotic substitute because of its advantages of safety, high efficiency and strong specificity. However, the mechanism of the action of IgY has not yet been clarified, and IgY can regulate the immune system of the animal body at the same time of inhibiting the pathogenic bacteria, which is to be further studied. The effects of IgY on various T-lymphocytes in the intestinal mucosa of mice challenged with Salmonella typhimurium were analyzed by in-vivo experiments. In addition, the effect of IgY on the phagocytosis of macrophages and the maturation of dendritic cells was also investigated by in vitro experiments. The effect of IgY on the various T-lymphocytes in the intestinal mucosa of the challenge mice was: Kunming mice were randomly divided into 4 groups, and the mice were pre-treated with streptomycin for 2 days and 12 hours, and the PBS, non-specific IgY and specific IgY were fed into the mice for 3 days, and the state of the mice was observed during this period. Mortality and body weight. After 3 days, the mice were dissected and the small intestine was taken, the lymph nodes of the intestinal tract, the epithelial layer and the lamina propria were isolated, and the number of the various lymphocytes was detected by flow cytometry. The results showed that after the challenge of Salmonella typhimurium, the state of mental state of mice was low, the death rate reached 60%, the body weight was significantly reduced, the enlargement of the Paar's lymph nodes, CD3 + T cells and CD8 + T cells were increased by 10% and 14%, respectively. There was a significant increase in the number of lymphocytes in the epithelium, and the CD8 + T cells and the TCR and the T cells increased by 18% and 16%, respectively. CD4 + T cells and CD8 + T cells increased by 8% and 12% in the native layer. After oral specific IgY, the state of mental state of the mice improved, the death rate was reduced to 20%, but the body weight did not change significantly. The lymph node enlargement was improved and the total number of T cells (CD3 + T cells) in the Paar lymph node was reduced by 6.5%, which was substantially restored to a normal level. The specific IgY can also significantly reduce the proportion of CD8 + T cells and TCR and T cells in the epithelial layer, which are respectively reduced by 12% and 10%, respectively. The lymphocytes in the intrinsic layer were also significantly reduced under specific IgY, in which the CD4 + T cells and CD8 + T cells were reduced by 7% and 12%, respectively. This indicates that the specific IgY can relieve the inflammatory reaction caused by the challenge of Salmonella typhimurium, and play a protective role on the body. The effect of IgY on the phagocytic activity of macrophages: The phagocytosis rate of macrophages and the release of NO were detected with FITC-labeled Salmonella typhimurium and then co-cultured with bacteria for 3 h. The results showed that the non-specific IgY and specific IgY could significantly increase the phagocytic rate of the macrophages to the bacteria by 20% and 33%, respectively. And the specific IgY obviously reduced the secretion of the macrophage NO, and the secretion amount of the NO was reduced to 14. m u.mol/ L from the original 26. m u.mol/ L. This suggests that IgY enhances the phagocytic rate of macrophages to bacteria and reduces the secretion of inflammatory factors. The effect of IgY on the maturation of dendritic cells: the differentiation of primary cells from the bone marrow of mice, the induction of differentiation into dendritic cells with 10 ng/ mL of rGM-CSF and rIL-4, and the collection of immature dendritic cells on day 7. Salmonella typhimurium was incubated with immature dendritic cells and specific IgY for 3 h, and the surface markers of dendritic cells were detected by flow cytometry. The results showed that the surface marker MHC II, CD40, CD80, CD83 and CD86 decreased by 11%,3%,4%,5% and 5%, respectively, while the specific IgY increased by 20%,9%,11%,15% and 5%, respectively. This indicates that after challenge, Salmonella typhimurium induced apoptosis of the dendritic cells, and after the addition of specific IgY, the apoptosis of the cells can be reduced and the maturation of the dendritic cells can be promoted.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.3
本文编号：2501589
[Abstract]:When the animal body is invaded by foreign pathogenic bacteria, the immune system is activated to resist the harm of the pathogenic bacteria to the body. However, the high activation of the immune system can lead to the immune stress phenomena such as the feed intake and the weight loss of the animals, which causes great economic losses to the breeding industry. So how to alleviate the immune stress has very important practical significance. The antibiotic can effectively improve the growth condition of the animal, but with the problems of drug residue and bacterial drug resistance in the animal product, the invention urgently needs to find a new product which not only can effectively control the disease of the culture animals, but also does not bring food safety problems and environmental hidden dangers to replace the antibiotics. The research shows that the yolk antibody can be used for preventing and treating infectious diseases of animals, and is considered to be the most promising new generation of green and safe antibiotic substitute because of its advantages of safety, high efficiency and strong specificity. However, the mechanism of the action of IgY has not yet been clarified, and IgY can regulate the immune system of the animal body at the same time of inhibiting the pathogenic bacteria, which is to be further studied. The effects of IgY on various T-lymphocytes in the intestinal mucosa of mice challenged with Salmonella typhimurium were analyzed by in-vivo experiments. In addition, the effect of IgY on the phagocytosis of macrophages and the maturation of dendritic cells was also investigated by in vitro experiments. The effect of IgY on the various T-lymphocytes in the intestinal mucosa of the challenge mice was: Kunming mice were randomly divided into 4 groups, and the mice were pre-treated with streptomycin for 2 days and 12 hours, and the PBS, non-specific IgY and specific IgY were fed into the mice for 3 days, and the state of the mice was observed during this period. Mortality and body weight. After 3 days, the mice were dissected and the small intestine was taken, the lymph nodes of the intestinal tract, the epithelial layer and the lamina propria were isolated, and the number of the various lymphocytes was detected by flow cytometry. The results showed that after the challenge of Salmonella typhimurium, the state of mental state of mice was low, the death rate reached 60%, the body weight was significantly reduced, the enlargement of the Paar's lymph nodes, CD3 + T cells and CD8 + T cells were increased by 10% and 14%, respectively. There was a significant increase in the number of lymphocytes in the epithelium, and the CD8 + T cells and the TCR and the T cells increased by 18% and 16%, respectively. CD4 + T cells and CD8 + T cells increased by 8% and 12% in the native layer. After oral specific IgY, the state of mental state of the mice improved, the death rate was reduced to 20%, but the body weight did not change significantly. The lymph node enlargement was improved and the total number of T cells (CD3 + T cells) in the Paar lymph node was reduced by 6.5%, which was substantially restored to a normal level. The specific IgY can also significantly reduce the proportion of CD8 + T cells and TCR and T cells in the epithelial layer, which are respectively reduced by 12% and 10%, respectively. The lymphocytes in the intrinsic layer were also significantly reduced under specific IgY, in which the CD4 + T cells and CD8 + T cells were reduced by 7% and 12%, respectively. This indicates that the specific IgY can relieve the inflammatory reaction caused by the challenge of Salmonella typhimurium, and play a protective role on the body. The effect of IgY on the phagocytic activity of macrophages: The phagocytosis rate of macrophages and the release of NO were detected with FITC-labeled Salmonella typhimurium and then co-cultured with bacteria for 3 h. The results showed that the non-specific IgY and specific IgY could significantly increase the phagocytic rate of the macrophages to the bacteria by 20% and 33%, respectively. And the specific IgY obviously reduced the secretion of the macrophage NO, and the secretion amount of the NO was reduced to 14. m u.mol/ L from the original 26. m u.mol/ L. This suggests that IgY enhances the phagocytic rate of macrophages to bacteria and reduces the secretion of inflammatory factors. The effect of IgY on the maturation of dendritic cells: the differentiation of primary cells from the bone marrow of mice, the induction of differentiation into dendritic cells with 10 ng/ mL of rGM-CSF and rIL-4, and the collection of immature dendritic cells on day 7. Salmonella typhimurium was incubated with immature dendritic cells and specific IgY for 3 h, and the surface markers of dendritic cells were detected by flow cytometry. The results showed that the surface marker MHC II, CD40, CD80, CD83 and CD86 decreased by 11%,3%,4%,5% and 5%, respectively, while the specific IgY increased by 20%,9%,11%,15% and 5%, respectively. This indicates that after challenge, Salmonella typhimurium induced apoptosis of the dendritic cells, and after the addition of specific IgY, the apoptosis of the cells can be reduced and the maturation of the dendritic cells can be promoted.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.3
【参考文献】
相关期刊论文 前1条
1 杨德胜;张险朋;黄炳炽;洪伟彬;;动物产品沙门氏菌污染情况调查[J];中国畜牧兽医;2010年10期
，本文编号：2501589
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2501589.html
