利用动物模型研究食物潜在致敏性及其致敏机理
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摘要
食物过敏发病率的逐年升高使其成为一个国际研究的热点。近年来人们一直在探索能够模拟人体食物致敏的理想动物致敏模型,以评价包括转基因食品在内的多种新型食品的潜在致敏性,并用于深入研究食物致敏的相关机理。目前,人们大都是针对单一食物致敏蛋白(如卵清白蛋白和花生凝集素等)进行动物致敏研究,而忽略了食物中多种蛋白间的互作对其致敏性的影响。另外,目前也缺乏对区分不同食物潜在致敏性的动物致敏方法的研究。本文在查阅相关文献并分析不同食物潜在致敏性的基础上,选择不同致敏性的花生蛋白提取物(PPE)、牛奶蛋白提取物(CME)和马铃薯蛋白提取物(PAPE)致敏BALB/c小鼠,研究了一种可以区分这些食物潜在致敏性适宜的小鼠致敏模型。在此基础上,采用PPE经口致敏小鼠,运用数字化表达谱分析(DGE)探究了小鼠致敏反应产生的机理及anti-CD4单克隆抗体对致敏反应产生的影响及其机制。主要研究结果及结论如下：
(1)使用PPE)、CME和PAPE分别经口致敏BALB/c小鼠,PPE使动物产生了最强烈的特异性IgE反应,最高水平的Thl-、Th2-细胞分化和Th2型细胞因子IL-4,还诱发肥大细胞在脾脏产生了最大程度的聚集和脱颗粒；CME处理组动物产生的各种免疫反应水平均显著低于PPE组(P<0.05); PAPE几乎未诱发小鼠产生致敏反应。说明本研究中选择的BALB/c小鼠及采用的致敏方法可以用以区分这些不同致敏性程度的食物总蛋白的潜在致敏性。
(2)使用DNAStar软件和NetMHC Ⅱ2.2Server在线服务器分析三种食物蛋白提取物的潜在致敏性。PPE中含有十一种已知致敏蛋白,其中Ara h1、Ara h2、Ara h3和Arah5具有较多的抗原表位；CME中含有三种已知致敏蛋白,其中仅B-乳球蛋白含有抗原表位；PAPE中含有四种已知致敏蛋白,但几乎都不含有抗原表位。模拟胃液消化实验中,PPE含有分子量分别为17KD(Arah2和／或Ara h8)和7KD的抗模拟胃液消化的蛋白或肽段,CME中只有B-乳球蛋白具有抗消化能力,PAPE被完全消化。这说明三种食物蛋白提取物潜在致敏性程度为PPE>CME> PAPE,进一步验证了使用BALB/c小鼠致敏模型的研究结果。
(3)经口给予C3H/HeJ小鼠PPE进行致敏,使用DGE方法分析致敏动物脾脏所有基因转录水平的变化以探究食物致敏过程相关分子机制。共鉴定出81个差异表达基因,其中有30个与免疫系统相关。这些基因主要涉及抗原呈递作用的抑制、免疫细胞迁移和粘附、T细胞激活信号的促进及B细胞受体(BCR)信号的增强。说明PPE处理激活了动物的获得性免疫反应。
(4)在PPE经口致敏C3H/HeJ小鼠的同时给予anti-CD4抗体,发现其从体液免疫、细胞免疫和全身系统性致敏症状三个方面抑制了动物过敏的产生。使用DGE方法进一步探索其影响过敏反应的机理,鉴定出131个差异表达基因,其中35个与免疫系统相关,它们主要涉及到机体巨噬细胞活化及固有免疫反应、T细胞和B细胞成熟、增殖和分泌抗体的抑制、以及特异性Treg细胞的促进等。说明anti-CD4抗体不仅抑制了动物对PPE的获得性免疫反应,还促进了机体特异性耐受及适度固有免疫反应的产生。
The increasing incidence make food allergy become an international research hotspot. In recent years, people have been exploring to ideal animal model of sensitization to evaluate many kinds of new food allergenic potential, and to study the related mechanism of food sensitization. However, at present, people are mostly research for single food sensitization protein (such as OVA, PNA) sensitization with animal, while ignoring the effects of proteins' mutual interaction in food on allergenicity. In this research, we chose three kind of food protein extracts from peanut, milk and potato to sensitize BALB/c mice and studied an allergic murine model that can recognize potential allergenicity of different food. And on this basis, peanut protein extract were used to oral administrate C3H/HeJ mice whose immune system is more sensitive. The digital gene expression analysis method (DGE) was then used to investigate the sensitization mechanism and the influence of non-depletion anti-CD4monoclonal antibody on the allergy reaction. The main results and conclusions are as follows:
(1) The BALB/c mice were orally sensitized with PPE, CME and PAPE respectively. Animals in PPE treatment group produced highest specific IgE, Thl-and Th2-cell, also the most severe aggregation and degranulation of mast cells in spleen. The levels of immune reaction of mice in CME group were lower than that in PPE group. The PAPE almost did not induce sensitization response in mice. This suggested that the chosen BALB/c mouse and sensitization method can be used to distinguish the different levels of potential allergenicity of food protein extracts.
(2) DNAStar and NetMHCⅡ2.2online Server were used to analyse and predict the potential allergenicity of PPE, CME and PAPE. There are11kind of known allergic proteins in PPE, the Ara h1, Ara h2, Ara h3and Ara h5contain much epitopes. CME contains three known sensitization protein and epitopes only exist in the β-LG. On the contract, there is no epitopes in the four known sensitized protein in PAPE. The results of simulated gastric fluid digestion experiment suggest that the molecular weight of anti-digestion allergen in PPE were17KD and7KD,β-LG is the only anti-digested protein in CME, and the PAPE was completely digested. These results demonstrated that the potential allergenicity of the three protein extracts was PPE> CME> PAPE, this further validate the results obtained from BALB/c mice allergic experiment.
(3) Orally sensitize C3H/HeJ mouse with PPE and explore the allergic related mechanism on molecular level using DGE method. The results appeared81differentially expressed genes were identified and contained30genes that are associated with immune system. These genes are mainly related to the inhibition of antigen-presenting function, immune cell migration and adhesion, promotion of T cell activation signal and enhancement of the B cell receptor (BCR) signal. This suggested that PPE processing activates the adoptive immune response of animal.
(4) Non-depletion anti-CD4monoclonal antibody was used to i.v treated C3H/HeJ mice at the same time of sensitization. We fund it inhibited the allergy reactions on three aspects of humoral immunity, cellular immunity and systemic symptoms. DGE was also used to investigate the mechanisms of this affluence.131differentially expressed genes containing35related to immune system were identified. The35genes were primarily involve of macrophage activation and the innate immune response, T cells and B cells mature, inhibition of proliferation and antibody production, and to promotion of specific Treg cells. Anti-CD4antibodies not only inhibit the adoptive immune responses of animals to the PPE, also promoted the specific tolerance and moderation of innate immune response.
(1)使用PPE)、CME和PAPE分别经口致敏BALB/c小鼠,PPE使动物产生了最强烈的特异性IgE反应,最高水平的Thl-、Th2-细胞分化和Th2型细胞因子IL-4,还诱发肥大细胞在脾脏产生了最大程度的聚集和脱颗粒；CME处理组动物产生的各种免疫反应水平均显著低于PPE组(P<0.05); PAPE几乎未诱发小鼠产生致敏反应。说明本研究中选择的BALB/c小鼠及采用的致敏方法可以用以区分这些不同致敏性程度的食物总蛋白的潜在致敏性。
(2)使用DNAStar软件和NetMHC Ⅱ2.2Server在线服务器分析三种食物蛋白提取物的潜在致敏性。PPE中含有十一种已知致敏蛋白,其中Ara h1、Ara h2、Ara h3和Arah5具有较多的抗原表位；CME中含有三种已知致敏蛋白,其中仅B-乳球蛋白含有抗原表位；PAPE中含有四种已知致敏蛋白,但几乎都不含有抗原表位。模拟胃液消化实验中,PPE含有分子量分别为17KD(Arah2和／或Ara h8)和7KD的抗模拟胃液消化的蛋白或肽段,CME中只有B-乳球蛋白具有抗消化能力,PAPE被完全消化。这说明三种食物蛋白提取物潜在致敏性程度为PPE>CME> PAPE,进一步验证了使用BALB/c小鼠致敏模型的研究结果。
(3)经口给予C3H/HeJ小鼠PPE进行致敏,使用DGE方法分析致敏动物脾脏所有基因转录水平的变化以探究食物致敏过程相关分子机制。共鉴定出81个差异表达基因,其中有30个与免疫系统相关。这些基因主要涉及抗原呈递作用的抑制、免疫细胞迁移和粘附、T细胞激活信号的促进及B细胞受体(BCR)信号的增强。说明PPE处理激活了动物的获得性免疫反应。
(4)在PPE经口致敏C3H/HeJ小鼠的同时给予anti-CD4抗体,发现其从体液免疫、细胞免疫和全身系统性致敏症状三个方面抑制了动物过敏的产生。使用DGE方法进一步探索其影响过敏反应的机理,鉴定出131个差异表达基因,其中35个与免疫系统相关,它们主要涉及到机体巨噬细胞活化及固有免疫反应、T细胞和B细胞成熟、增殖和分泌抗体的抑制、以及特异性Treg细胞的促进等。说明anti-CD4抗体不仅抑制了动物对PPE的获得性免疫反应,还促进了机体特异性耐受及适度固有免疫反应的产生。
The increasing incidence make food allergy become an international research hotspot. In recent years, people have been exploring to ideal animal model of sensitization to evaluate many kinds of new food allergenic potential, and to study the related mechanism of food sensitization. However, at present, people are mostly research for single food sensitization protein (such as OVA, PNA) sensitization with animal, while ignoring the effects of proteins' mutual interaction in food on allergenicity. In this research, we chose three kind of food protein extracts from peanut, milk and potato to sensitize BALB/c mice and studied an allergic murine model that can recognize potential allergenicity of different food. And on this basis, peanut protein extract were used to oral administrate C3H/HeJ mice whose immune system is more sensitive. The digital gene expression analysis method (DGE) was then used to investigate the sensitization mechanism and the influence of non-depletion anti-CD4monoclonal antibody on the allergy reaction. The main results and conclusions are as follows:
(1) The BALB/c mice were orally sensitized with PPE, CME and PAPE respectively. Animals in PPE treatment group produced highest specific IgE, Thl-and Th2-cell, also the most severe aggregation and degranulation of mast cells in spleen. The levels of immune reaction of mice in CME group were lower than that in PPE group. The PAPE almost did not induce sensitization response in mice. This suggested that the chosen BALB/c mouse and sensitization method can be used to distinguish the different levels of potential allergenicity of food protein extracts.
(2) DNAStar and NetMHCⅡ2.2online Server were used to analyse and predict the potential allergenicity of PPE, CME and PAPE. There are11kind of known allergic proteins in PPE, the Ara h1, Ara h2, Ara h3and Ara h5contain much epitopes. CME contains three known sensitization protein and epitopes only exist in the β-LG. On the contract, there is no epitopes in the four known sensitized protein in PAPE. The results of simulated gastric fluid digestion experiment suggest that the molecular weight of anti-digestion allergen in PPE were17KD and7KD,β-LG is the only anti-digested protein in CME, and the PAPE was completely digested. These results demonstrated that the potential allergenicity of the three protein extracts was PPE> CME> PAPE, this further validate the results obtained from BALB/c mice allergic experiment.
(3) Orally sensitize C3H/HeJ mouse with PPE and explore the allergic related mechanism on molecular level using DGE method. The results appeared81differentially expressed genes were identified and contained30genes that are associated with immune system. These genes are mainly related to the inhibition of antigen-presenting function, immune cell migration and adhesion, promotion of T cell activation signal and enhancement of the B cell receptor (BCR) signal. This suggested that PPE processing activates the adoptive immune response of animal.
(4) Non-depletion anti-CD4monoclonal antibody was used to i.v treated C3H/HeJ mice at the same time of sensitization. We fund it inhibited the allergy reactions on three aspects of humoral immunity, cellular immunity and systemic symptoms. DGE was also used to investigate the mechanisms of this affluence.131differentially expressed genes containing35related to immune system were identified. The35genes were primarily involve of macrophage activation and the innate immune response, T cells and B cells mature, inhibition of proliferation and antibody production, and to promotion of specific Treg cells. Anti-CD4antibodies not only inhibit the adoptive immune responses of animals to the PPE, also promoted the specific tolerance and moderation of innate immune response.
引文
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