【摘要】：當動物機體受到外來病原菌入侵時,免疫系統(tǒng)被激活以抵御病原菌對機體的傷害。然而免疫系統(tǒng)的高度活化會導致動物采食量及體重下降等免疫應(yīng)激現(xiàn)象,這給養(yǎng)殖業(yè)造成巨大經(jīng)濟損失。因此如何緩解免疫應(yīng)激具有非常重要的實際意義。抗生素可有效改善動物的生長狀況,但隨著動物產(chǎn)品中藥物殘留和細菌耐藥性等問題的出現(xiàn),迫切需要尋找一種既能有效控制養(yǎng)殖動物疾病,又不帶來食品安全問題和環(huán)境隱患的新產(chǎn)品以替代抗生素。研究表明,卵黃抗體可用于動物感染性疾病的防治,因其具有安全、高效、特異性強等優(yōu)點,所以被認為是最有前景的新一代綠色、安全型抗生素替代品。然而,關(guān)于IgY的作用機理現(xiàn)在還未闡明,IgY在抑制病原菌的同時,能否對動物機體的免疫系統(tǒng)起到調(diào)節(jié)作用,有待于進一步的研究。本課題在前期研究的基礎(chǔ)上,通過體內(nèi)實驗分析IgY對鼠傷寒沙門氏菌攻毒小鼠腸道黏膜各種T淋巴細胞的影響。此外,通過體外實驗還考察了IgY對巨噬細胞吞噬活性和樹突細胞成熟度的影響。IgY對攻毒小鼠腸道黏膜各種T淋巴細胞的影響：昆明小鼠隨機分為4組,用鏈霉素預處理2d,12h后灌胃PBS、非特異性IgY和特異性IgY,持續(xù)3 d,在此期間觀察小鼠的狀態(tài)、死亡率和體重。3d后解剖小鼠并取小腸,分離腸道派爾淋巴結(jié)、上皮層以及固有層的淋巴細胞,用流式細胞儀檢測各種淋巴細胞的數(shù)量變化。結(jié)果表明,鼠傷寒沙門氏菌攻毒后,小鼠精神狀態(tài)萎靡,死亡率達到60%,體重明顯減輕,派爾淋巴結(jié)出現(xiàn)腫大,CD3+T細胞和CD8+T細胞分別提高10%和14%。上皮內(nèi)淋巴細胞出現(xiàn)大量增加,CD8+T細胞和TCRγδ+T細胞分別增加18%和16%。固有層中CD4+T細胞和CD8+T細胞升高8%和12%�？诜禺愋訧gY后,小鼠精神狀態(tài)好轉(zhuǎn),死亡率降至20%,但體重無明顯變化。派爾淋巴結(jié)腫大狀況好轉(zhuǎn),并且派爾淋巴結(jié)中總的T細胞(CD3+T細胞)降低6.5%,基本恢復到正常水平。特異性IgY還可顯著降低上皮層中CD8+T細胞和TCRγδ+T細胞的比例,分別降低12%和10%。固有層中的淋巴細胞在特異性IgY的作用下,也顯著降低,其中CD4+T細胞和CD8+T細胞分別減少7%和12%。這表明,特異性IgY可緩解鼠傷寒沙門氏菌攻毒引起的炎癥反應(yīng),對機體起到保護作用。IgY對巨噬細胞吞噬活性的影響：用FITC標記鼠傷寒沙門氏菌,然后巨噬細胞與細菌共培養(yǎng)3h后,檢測巨噬細胞的吞噬率及NO的釋放量。結(jié)果表明,非特異性IgY和特異性IgY可顯著提高巨噬細胞對細菌的吞噬率,分別提高20%和33%。并且特異性IgY顯著減少了巨噬細胞NO的分泌,使NO的分泌量由原先的26μmol/L降低至14 μmol/L.這表明IgY可增強巨噬細胞對細菌的吞噬率并減少炎癥因子的分泌。IgY對樹突細胞成熟度的影響：從小鼠骨髓中分離原代細胞,用10 ng/mL rGM-CSF和rIL-4誘導分化成樹突細胞,收集第7d的未成熟樹突細胞。將鼠傷寒沙門氏菌與未成熟樹突細胞以及特異性IgY共同培養(yǎng)3 h,用流式細胞儀檢測樹突細胞表面標志的變化。結(jié)果表明,鼠傷寒沙門氏菌攻毒后樹突細胞表面標志MHCⅡ, CD40, CD80, CD83和CD86分別降低11%、3%、4%、5%和5%,而加入特異性IgY后分別升高20%、9%、11%、15%和5%。這說明攻毒后,鼠傷寒沙門氏菌引起樹突細胞的凋亡,而加入特異性IgY后,可以減少細胞的凋亡并促進樹突細胞的成熟。
[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.
【學位授予單位】：大連理工大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.3

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相關(guān)期刊論文 前1條

1 楊德勝;張險朋;黃炳熾;洪偉彬;;動物產(chǎn)品沙門氏菌污染情況調(diào)查[J];中國畜牧獸醫(yī);2010年10期

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本文編號：2501589