特异性IgY对肠道感染小鼠黏膜免疫的影响

发布时间：2018-01-13 11:47

本文关键词：特异性IgY对肠道感染小鼠黏膜免疫的影响　出处：《大连理工大学》2011年硕士论文　论文类型：学位论文

【摘要】：卵黄抗体(Egg Yolk immunoglobulin, IgY)作为具有广阔前景的抗生素替代品,在防治感染性疾病的应用中取得了良好的效果,但对其作用机理的研究还处于初始阶段。本论文运用鼠伤寒沙门氏菌小鼠肠道感染模型,探讨特异性IgY对炎症反应中炎症因子的表达量的影响,考察特异性IgY与黏膜免疫系统的相互作用。本研究考察不同的菌种、小鼠品系、小鼠年龄、抗生素处理及攻毒剂量和持续时间对小鼠肠道感染模型建立的影响。结果表明使用5mg/ml链霉素处理2天后,3×109CFU/ml浓度鼠伤寒沙门氏菌灌胃攻毒4周龄昆明小鼠,连续2天,每天0.4 ml,可致小鼠痉挛、抽搐甚至死亡,死亡率可达60%以上,解剖可观察到肠道黏膜充血肿胀。以上症状明显且可重复,证实模型建立成功。本研究采用灭活的鼠伤寒沙门氏全菌作为抗原免疫蛋鸡制备特异性抗体。采用水稀释-盐析-超滤技术从卵黄中分离纯化IgY,抗体纯度可达90%,回收率为90.6mg/egg。ELISA检测可知,免疫第6周的抗体效价达到最大值,为1：10000。免疫荧光实验表明,病原菌与特异性IgY特异性结合,与非特异性IgY存在部分结合。抑菌实验表明特异性IgY在液体培养基中以剂量依赖方式抑制病原菌的生长,IgY浓度为20 mg/ml时抑菌效果明显。以0.4ml 3×109CFU/ml浓度的菌液灌胃小鼠后,再分别给小鼠灌胃PBS、20 mg/ml特异性IgY、20 mg/ml非特异性IgY和5 mg/ml链霉素溶液,观察10天内小鼠状态和死亡率。攻毒后第5天解剖小鼠,取肠道组织,提取总RNA,用Real-time PCR方法检测肠道组织中炎症因子(IFN-γ、IL-6和IL-10)的表达水平。结果表明,特异性IgY可有效降低鼠伤寒沙门氏菌感染造成的小鼠病症和死亡率,改善肠道病理状态,且小鼠肠道中促炎因子IFN-γ的表达量显著降低,抗炎因子IL-10表达量显著增加(P0.05)。说明特异性IgY可以降低小鼠肠道感染时的炎症反应。同时IgY还可以增加肠道中IL-6的表达量,该细胞因子具有促进抗体分泌的作用。本研究建立了昆明小鼠的鼠伤寒沙门氏菌肠道感染模型,制备了抗鼠伤寒沙门氏菌的特异性IgY,证实了特异性IgY可降低鼠伤寒沙门氏菌感染造成的死亡率。特异性IgY可以降低促炎因子和提高抗炎因子的表达量,减轻肠道病理症状,降低炎症反应。研究结果为特异性IgY防治感染性疾病机理提供了新的研究方向。
[Abstract]:Yolk antibody Egg Yolk immunoglobulin (IgY) as a promising alternative to antibiotics. Good results have been achieved in the application of prevention and treatment of infectious diseases, but the study of its mechanism is still in the initial stage. In this paper, the intestinal infection model of mice with Salmonella typhimurium was used. To investigate the effect of specific IgY on the expression of inflammatory factors in inflammatory response and the interaction between specific IgY and mucosal immune system. In this study, different strains, strains and ages of mice were investigated. Effects of antibiotic treatment, dose and duration on the establishment of intestinal infection model in mice. The results showed that 5 mg / ml streptomycin was used for 2 days. 3 脳 10 9 CFU / ml mice were injected with Salmonella typhimurium by stomach for 4 weeks. For 2 days, 0.4 ml per day could induce convulsions, convulsions and even death in mice. The mortality rate was more than 60%, the intestinal mucosal congestion and swelling could be observed by anatomy, and the above symptoms were obvious and repeatable. It was proved that the model was established successfully. In this study, the inactivated Salmonella typhimurium was used as antigen to immunize laying hens to prepare specific antibody. The purified IgY was purified from egg yolk by water dilution, salting-out and ultrafiltration, and the purity of the antibody was up to 90%. The recovery rate of 90.6 mg / egg.ELISA showed that the titer of antibody reached the maximum of 1: 10000in the 6th week of immunization. The immunofluorescence test showed that the antibody titer was 1: 10000. The bacteriostasis experiment showed that the specific IgY inhibited the growth of the pathogen in a dose-dependent manner in a dose-dependent manner. When the concentration of IgY was 20 mg/ml, the bacteriostatic effect was obvious. Mice were gavaged with 0.4 ml 3 脳 10 9 CFU / ml bacterial solution, and then 20 mg/ml specific IgY were given to mice. 20 mg/ml nonspecific IgY and 5 mg/ml streptomycin solution were used to observe the state and mortality of mice within 10 days. The expression of IFN- 纬 -IL-6 and IL-10 in intestinal tissue was detected by Real-time PCR method. Specific IgY can effectively reduce the disease and mortality of mice caused by Salmonella typhimurium infection, improve the intestinal pathological state, and the expression of pro-inflammatory factor IFN- 纬 in the intestinal tract of mice decreased significantly. The expression of anti-inflammatory factor IL-10 increased significantly (P0.05). The results showed that specific IgY could decrease the inflammatory response of mice with intestinal infection, and IgY could increase the expression of IL-6 in the intestine. The cytokine has the function of promoting antibody secretion. In this study, the model of intestinal infection of Salmonella typhimurium in Kunming mice was established, and the specific IgY against Salmonella typhimurium was prepared. It is confirmed that specific IgY can reduce the mortality caused by Salmonella typhimurium infection, and specific IgY can reduce the expression of proinflammatory factor and anti-inflammatory factor, and alleviate the intestinal pathological symptoms. The results provide a new research direction for the mechanism of specific IgY in the prevention and treatment of infectious diseases.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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