【作者】 刘潇；

【导师】 杨正涛；

【作者基本信息】 吉林大学 ， 临床兽医学， 2020， 硕士

【摘要】 大豆中的营养丰富,不但必须氨基酸含量高,而且氨基酸也较为平衡,因此一直以来大豆都是动物优质的植物蛋白源,但其中存在的抗营养因子如α-伴大豆球蛋白(α-conglycinin)、β-伴大豆球蛋白(β-conglycinin)、大豆球蛋白(Glycinin)等可通过激活中性粒细胞发生先天免疫应答反应从而使仔猪等幼龄动物出现肠道过敏症状,给畜牧养殖业造成经济损失,解决伴大豆球蛋白这一类抗营养因子对幼畜健康的危害需要首先阐明α、β-伴大豆球蛋白损伤幼龄动物肠道黏膜的详细机制。作为机体的第一道防线,肠道中的中性粒细胞在接触到抗原后,可通过吞噬及脱颗粒等应答反应对其进行降解,而近年来又发现中性粒细胞活化后可以释放中性粒细胞胞外诱捕网(neutrophilextracellular traps,NETs)参与抗原的免疫应答反应。NETs是把双刃剑,除了可以捕杀抗原物质之外,也参与多种疾病的病理生理过程,当NETs形成过多或者不能被及时降解,可能对自身细胞及组织造成免疫损伤。大量的研究报道证实了大豆中的抗原蛋白可以引起肠道系统的免疫应答,然而大豆中的抗原蛋白如α、β-伴大豆球蛋白对中性粒细胞产生NETs的影响以及可能引起的后果尚未见报道。本论文首先进行体外实验,用α-伴大豆球蛋白和β-伴大豆球蛋白作用于小鼠的中性粒细胞,观察和验证α-伴大豆球蛋白和β-伴大豆球蛋白对NETs生成的影响。进而通过体内实验,选取β-伴大豆球蛋白对小鼠进行灌胃,构建β-伴大豆球蛋白小鼠腹泻过敏动物模型。旨在为生产实践中有效提高豆粨的利用价值以及降低仔幼龄动物肠道疾病的发病率而提供初步的研究依据及理论基础。首先,采取小鼠的血液,用密度梯度离心法获得中性粒细胞,利用激光共聚焦显微镜和western blotting等技术,探究α、β-伴大豆球蛋白对NETs形成的影响及其潜在的机制。结果表明,α、β-伴大豆球蛋白能够诱导以DNA为骨架,其间固定弹性蛋白酶、髓过氧化物酶等蛋白颗粒的NETs的释放。其机制可能是依赖于增加活性氧生成、MAPK信号转导通路ERK1/2和p38表达的激活。其次,选取β-伴大豆球蛋白建立小鼠腹泻过敏动物模型,制作组织病理学切片,Pico~?Green检验血清中NETs的含量,ELISA检测血清中IgE的水平,初步评估β-伴大豆球蛋白对小鼠过敏反应的影响。组织病理学结果显示,β-伴大豆球蛋白组的小鼠小肠绒毛上皮细胞出现局部局灶性坏死,小肠绒毛的固有层出现淋巴细胞、中性粒细胞等炎性细胞浸润,DNaseⅠ预处理可以降低β-伴大豆球蛋白引起的组织病理学损伤;NETs定量结果显示β-伴大豆球蛋白显著增加了血清中NETs的含量,而生成的NETs可以被DNaseⅠ处理降解,说明腹泻小鼠的血清中可以检测到NETs;ELISA结果表明β-伴大豆球蛋白引起小鼠血清中的IgE水平显著升高,DNaseⅠ预处理可以降低β-伴大豆球蛋白引起的IgE水平的升高。以上结果表明肠道免疫系统中的NETs水平与肠道组织损伤程度密切相关。通过以上研究,本论文初步证实了α、β-伴大豆球蛋白通过激活ROS的产生和MAPK信号转导通路ERK1/2和p38蛋白的表达等诱导小鼠中性粒细胞产生NETs。并建立了小鼠过敏模型,探究NETs对β-伴大豆球蛋白引起的肠道先天免疫系统的影响,从而为幼龄动物肠道过敏的预防和治疗提供理论依据和指导。更多还原

【Abstract】 Soybean is rich in nutrition,not only must have high amino acid content,but also the amino acid is relatively balanced,so soybean and tempeh have been high-quality plant protein sources for livestock and poultry,but anti-nutritional factors such as α-conglycinin,β-conglycinin,Glycinin,etc.can activate the neutrophil to generate an innate immune response to cause intestinal allergic symptoms in young animals such as piglets and give livestock The aquaculture industry has caused economic losses.To address the harm of anti-nutritional factors such as conglycinin to the health of young animals,the detailed mechanism of α,β-conglycinin damage to the intestinal mucosa of young animals must first be clarified.As the body’s first line of defense,neutrophils in the intestine can be degraded by response reactions such as phagocytosis and degranulation after contact with the antigen.In recent years,it has been found that neutrophils can form neutrophils after activation.The extracellular structures of extracellular traps(NETs)are involved in the immune response of the antigen.Studies have shown that NETs is a double-edged sword.In addition to killing pathogens,it also participates in the pathophysiology of many diseases.When NETs are formed too much or cannot be degraded in time,they may cause immune damage to their cells and tissues.A large number of research reports have confirmed that soy antigen proteins can cause an immune response in the intestinal system.However,the effects of antigenic proteins such as α,β-conglycinin on NETs produced by neutrophils and the possible consequences have not been reported.In this paper,in vitro experiments were conducted to observe and verify the effects of alpha-alpha-soy globulin and alpha-alpha-soy globulin on the neutrophils of mice.Furthermore,through in vivo experiments,β-conglycinin was selected for intragastric administration of mice,and a β-conglycinin mouse diarrhea allergy animal model was constructed.The aim is to provide preliminary research basis and theoretical basis for effectively increasing the utilization value of tempeh in production practices and reducing the incidence of intestinal diseases in young animals.Firstly,neutrophils were obtained from the blood of mice by density gradient centrifugation.The effects of α and β-conglycinin on the formation of NETs and their underlying mechanisms were investigated using laser confocal microscope and western blotting.The results show that α,β-conglycinin can induce the release of NETs with DNA as the backbone,and fix protein particles such as elastase and myeloperoxidase.The mechanism may depend on the increase of reactive oxygen species production,activation of MAPK signal transduction pathway ERK1 / 2 and p38 expression.Secondly,the animal model of mice diarrhea allergy was established by using estrogen-associated soybean globulin,and histopathological sections were prepared.The nucleic acid content in serum was tested by Pico~? Green,and the serum IgE level was detected by ELISA,so as to preliminarily evaluate the effect of estrogen-associated soybean globulin on diarrhea allergy in mice.Histopathological results showed that the small intestinal villous epithelial cells of the β-conglycinin group showed local focal necrosis,and the lamina propria of the small intestine showed infiltration of inflammatory cells such as lymphocytes and neutrophils,and DNase Ⅰ pretreatment.Can reduce the histopathological damage caused by β-conglycinin;nucleic acid results show that β-conglycinin significantly increases the content of nucleic acid in serum,and the generated nucleic acid can be degraded by DNase Ⅰ treatment,indicating that the serum of diarrhea mice NETs can be detected in ELISA;ELISA results showed that β-conglycinin caused a significant increase in IgE levels in mouse serum,and DNase I pretreatment could reduce the increase in IgE levels caused by β-conglycinin.The above results indicate that the level of NETs in the intestinal immune system is closely related to the degree of intestinal tissue damage.Based on the above studies,this paper has initially confirmed that α,β-conglycinin induces neutrophils to produce NETs in mice by activating ROS production and MAPK signal transduction pathway ERK1 / 2 and p38 protein expression.A mouse allergy model was established to investigate the effect of NETs on the intestinal innate immune system caused by β-conglycinin,so as to provide theoretical basis and guidance for the prevention and treatment of intestinal allergy in young animals.更多还原