本文關(guān)鍵詞：特異性IgY對腸道感染小鼠黏膜免疫的影響　出處：《大連理工大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 卵黃抗體 鼠傷寒沙門氏菌 腸道黏膜免疫 炎癥因子

【摘要】：卵黃抗體(Egg Yolk immunoglobulin, IgY)作為具有廣闊前景的抗生素替代品,在防治感染性疾病的應(yīng)用中取得了良好的效果,但對其作用機(jī)理的研究還處于初始階段。本論文運(yùn)用鼠傷寒沙門氏菌小鼠腸道感染模型,探討特異性IgY對炎癥反應(yīng)中炎癥因子的表達(dá)量的影響,考察特異性IgY與黏膜免疫系統(tǒng)的相互作用。 本研究考察不同的菌種、小鼠品系、小鼠年齡、抗生素處理及攻毒劑量和持續(xù)時(shí)間對小鼠腸道感染模型建立的影響。結(jié)果表明使用5mg/ml鏈霉素處理2天后,3×109CFU/ml濃度鼠傷寒沙門氏菌灌胃攻毒4周齡昆明小鼠,連續(xù)2天,每天0.4 ml,可致小鼠痙攣、抽搐甚至死亡,死亡率可達(dá)60%以上,解剖可觀察到腸道黏膜充血腫脹。以上癥狀明顯且可重復(fù),證實(shí)模型建立成功。 本研究采用滅活的鼠傷寒沙門氏全菌作為抗原免疫蛋雞制備特異性抗體。采用水稀釋-鹽析-超濾技術(shù)從卵黃中分離純化IgY,抗體純度可達(dá)90%,回收率為90.6mg/egg。ELISA檢測可知,免疫第6周的抗體效價(jià)達(dá)到最大值,為1：10000。免疫熒光實(shí)驗(yàn)表明,病原菌與特異性IgY特異性結(jié)合,與非特異性IgY存在部分結(jié)合。抑菌實(shí)驗(yàn)表明特異性IgY在液體培養(yǎng)基中以劑量依賴方式抑制病原菌的生長,IgY濃度為20 mg/ml時(shí)抑菌效果明顯。 以0.4ml 3×109CFU/ml濃度的菌液灌胃小鼠后,再分別給小鼠灌胃PBS、20 mg/ml特異性IgY、20 mg/ml非特異性IgY和5 mg/ml鏈霉素溶液,觀察10天內(nèi)小鼠狀態(tài)和死亡率。攻毒后第5天解剖小鼠,取腸道組織,提取總RNA,用Real-time PCR方法檢測腸道組織中炎癥因子(IFN-γ、IL-6和IL-10)的表達(dá)水平。結(jié)果表明,特異性IgY可有效降低鼠傷寒沙門氏菌感染造成的小鼠病癥和死亡率,改善腸道病理狀態(tài),且小鼠腸道中促炎因子IFN-γ的表達(dá)量顯著降低,抗炎因子IL-10表達(dá)量顯著增加(P0.05)。說明特異性IgY可以降低小鼠腸道感染時(shí)的炎癥反應(yīng)。同時(shí)IgY還可以增加腸道中IL-6的表達(dá)量,該細(xì)胞因子具有促進(jìn)抗體分泌的作用。 本研究建立了昆明小鼠的鼠傷寒沙門氏菌腸道感染模型,制備了抗鼠傷寒沙門氏菌的特異性IgY,證實(shí)了特異性IgY可降低鼠傷寒沙門氏菌感染造成的死亡率。特異性IgY可以降低促炎因子和提高抗炎因子的表達(dá)量,減輕腸道病理癥狀,降低炎癥反應(yīng)。研究結(jié)果為特異性IgY防治感染性疾病機(jī)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- 緯 -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- 緯 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.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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