植物源重组人血清白蛋白对BALB/c小鼠的致敏性评价

目的利用BALB/c小鼠,探讨植物源重组人血清白蛋白(oryza sativa recombinant human serum albumin,OsrHSA)是否具有潜在致敏性。方法将80只BALB/c小鼠按体重随机分为4组,分别为卵清蛋白阳性对照组、马铃薯酸性磷酸酶(potato acid phosphatase,PAP)阴性对照组、OsrHSA组和磷酸盐缓冲液溶剂对照组,每组20只。分别于第0、3、6、9和12 d给小鼠腹腔注射0.2 mg/mL相应蛋白溶液。酶联免疫法检测血浆组胺水平和血清中特异性IgE抗体(sIgE)、特异性IgG抗体(sIgG)、特异性IgG1抗体(sIgG1)、特异性IgG2a抗体(sIgG2a)及总IgE抗体(tIgE)水平。结果卵清蛋白组血清tIgE、sIgE、sIgG、sIgG1和血浆组胺含量显著高于其他各组,而血清sIgG2a含量降低,差异有统计学意义(P0.05)。OsrHSA组血清sIgE水平和组胺水平与溶剂对照组相比差异无统计学意义(P0.05),血清sIgG、sIgG1和sIgG2a含量低于PAP组(P0.05),而血清tIgE含量与PAP组相比差异无统计学意义(P0.05)。结论腹腔注射OsrHSA对BALB/c小鼠的潜在致敏性很低。     

蛋白过敏性研究——BN大鼠动物模型

目的　用BN大鼠模型探讨不同蛋白的过敏性。方法　通过腹腔注射 (1mg ml)或口服 (10mg ml)给予BN大鼠卵清蛋白 (OVA)或牛血清白蛋白 (BSA) ,时间为 6周。在不同时间取血清 ,进行被动皮肤致敏试验 (PCA) ;分离血浆进行组胺测定 ;此外 ,对OVA组和对照组的大鼠进行血压测定。结果　PCA试验结果表明OVA特异性IgE抗体滴度较高 (1 32～ 1 12 8) ,而BSA特异性IgE抗体滴度较低 (1 2～ 1 4 )。与对照组相比 ,腹腔注射或口服OVA均使组胺水平升高。口服OVA对大多数大鼠的血压没有明显的影响 ,然而 ,有 2只大鼠收缩压暂时降低。结论　该研究结果表明BN大鼠模型可能是一种较理想的蛋白过敏性模型。     

复方中药CRFC-1对花生食物过敏拮抗作用的研究

目的探讨复方中药CRFC-1在食物过敏实验性小鼠动物模型中拮抗花生诱导的食物变态反应效果及机制。方法以花生全蛋白提取物(WPE)和葡萄球菌肠毒素B(SEB)混合物致敏、激发建立食物过敏实验性小鼠动物模型,设立CRFC-1实验组和对照组。观察小鼠过敏症状,ELISA检测血清特异性IgE、IL-4、组胺水平,并做脱颗粒肥大细胞计数。结果 CRFC-1实验组小鼠过敏症状、血清特异性IgE、IL-4、组胺水平及脱颗粒肥大细胞数明显低于对照组,差异有统计学意义(P﹤0.05)。结论复方中药CRFC-1在小鼠动物模型中能够阻断WPE诱导的食物变态反应。     

食品致敏性评价啮齿类动物模型研究——C3H/HeJ小鼠动物模型

目的研究C3H/HeJ小鼠作为食品致敏性评价动物模型的可行性,探讨不同致敏途径适当的致敏周期以及辅助性T淋巴细胞因子的变化。方法通过经口灌胃及腹腔注射两种途径给予雌性C3H/HeJ小鼠致敏蛋白——卵清蛋白(OVA),共35天。分别在第14、28、35天内眦取血分离血清,测定OVA特异性IgE。于第35天取脾脏,荧光实时定量PCR检测Th2型细胞因子IL-4及Th1型细胞因子IFN-γ的mRNA表达水平。结果与阴性对照组相比,经口灌胃OVA组小鼠第28和35天血清中OVA特异性IgE浓度显著升高(P0.05);腹腔注射OVA+Al(OH)3组小鼠第14、28和35天血清中OVA特异性IgE浓度显著升高(P0.05)。与阴性对照组相比,OVA组与OVA+Al(OH)3组小鼠细胞因子IL-4的表达水平均显著升高;细胞因子IFN-γ的mRNA表达水平均显著降低。结论C3H/HeJ小鼠可以作为食品致敏性评价动物模型,经口灌胃途径及腹腔注射途径的最佳致敏周期分别为14天和28天。     

经口致敏食物过敏大鼠模型的建立

目的探讨经口致敏食物过敏大鼠模型的建立方法,以及适宜的评价指标。方法以卵清蛋白(OVA)作为致敏原,将16只3周龄Brown-Norway(BN)大鼠随机分为3组:阴性对照组(生理盐水灌胃)、阳性对照组(OVA腹腔注射组)和实验组(OVA灌胃组),共灌胃9周。在第4、5、6、7、8、9周分别采用双抗体夹心ELISA法及皮肤过敏实验法(PCA)测定实验动物血清OVA特异性IgE(OVA-IgE)抗体滴度,以判断动物是否致敏成功,在第13周各组动物给予100mg/mlOVA1ml灌胃激发后测血清OVA-IgE滴度。结果ELISA结果显示在灌胃第6周时实验组动物OVA-IgE滴度水平达到最高,且显著高于阴性对照组(P<0.05),致敏率为60%(3/5);第7、第8周时OVA-IgE滴度水平略有下降,但是仍然显著高于阴性对照组(P<0.05),致敏率达到80%(4/5),OVA-IgE水平和致敏率与阳性对照组之间差异无显著性;实验组PCA结果在第6、7、8周均显示为阴性,而阳性对照组PCA显示为阳性。结论经口致敏可作为一种可行的建立食物过敏动物模型的方法,符合食物过敏发生的自然生理过程,建议适宜致敏时间为6周;采用ELISA法检测血清OVA-IgE抗体水平较PCA法能更加灵敏地反映经口致敏模型是否成功。     

食物蛋白质消化稳定性和热稳定性研究

目的建立体外模拟胃肠液消化稳定性试验和热稳定性试验方法,确定试验中的阳性和阴性参考蛋白质。方法在体外模拟胃肠液消化环境和热加工处理环境,通过SDS-PAGE凝胶电泳研究常见食物致敏原(鸡卵清蛋白OVA、牛β-乳球蛋白β-LG)、不常见食物致敏原(牛血清白蛋白BSA)和非致敏原(大豆脂肪水解酶LPE、马铃薯酸性磷酸酶PAP)在其中各观察时间点的降解情况。结果OVA在模拟胃肠液中60min不降解;β-LG在模拟胃液中60min不降解稳定存在,但在模拟肠液中30min内完全降解;BSA在模拟胃液中30min内完全降解,在模拟肠液中60min部分降解;PAP在模拟胃液中15s即完全降解,LPE在30s内完全降解;而在模拟肠液中,PAP60min完全不降解,LPE60min大部分降解。在热稳定性试验中,OVA、β-LG、BSA60min内不降解,PAP60min内完全降解。结论模拟胃液消化稳定性和热稳定性试验中设OVA为常见致敏原对照、BSA为不常见致敏原对照、PAP为非致敏原对照,模拟肠液消化稳定性试验中设OVA为常见致敏原对照、BSA为不常见致敏原对照、LPE为非致敏原对照。并据此初步建立体外消化稳定性和热稳定性试验方法。     

金银花水提物与益生菌治疗致敏小鼠作用比较

[目的]比较中药金银花水提物(以下简称金银花)与双歧杆菌、乳酸杆菌治疗卵清蛋白(OVA)致敏小鼠免疫调节作用. [方法]BALB/c雌鼠40只均分为5组,按照肠道激发后灌服不同的药物随机分为金银花高(HLT100%)、中(MLT 50%)浓度组,益生菌组(PT),致敏对照(激发后生理盐水灌服),以及正常对照(生理盐水灌服).取空肠标本行HE及甲苯胺蓝染色;取腹腔巨噬细胞行瑞氏染色;平板活菌计数肠道双歧杆菌、乳酸杆菌;ELISA法检测血清OVA特异性IgE(sIgE)、sIgG2a及小肠粘液总IgA、sIgA水平. [结果]金银花和益生菌均可缓解致敏小鼠小肠炎症,改善肥大细胞聚集和脱颗粒,提高腹腔巨噬细胞吞噬率与吞噬指数;肠道双歧杆菌、乳酸杆菌增多,以HLT组显著;致敏小鼠血清sIgE下降,sIgG2a增加,致sIgE/sIgG2a比值下降,肠道总IgA、sIgA水平增加,以高浓度金银花作用更明显.[结论]首次比较研究金银花与益生菌治疗OVA介导的致敏小鼠速发型变态反应,证实金银花与益生菌均可明显改善致敏小鼠的过敏症状作用,高浓度金银花治疗效果更好.     

重组粉尘螨Ⅱ类变应原致敏小鼠肺部变应性炎症模型的建立

目的探讨以重组粉尘螨Ⅱ类变应原(rDerf2)建立小鼠肺部变应性炎症模型的方法。方法将30只6～8周龄雌性SPF级BALB/c小鼠,随机分为PBS组(阴性对照组)、卵清蛋白(OVA)组(阳性对照组)和rDerf2组(实验组)。实验组分别于第0、7、14天用rDerf2对小鼠腹腔进行注射致敏;再于第21天开始,连续7d进行雾化激发;对照组分别用PBS和OVA代替。最后一次雾化激发后24h内处死动物,观察肺组织病理变化、支气管肺泡灌洗液(BLAF)中的白细胞计数及分类,用ELISA测定BLAF和脾细胞培养上清中白细胞介素2(IL-2)、IL-4、IL-17和IFN-γ的含量及血清中IgG1、IgE抗体水平变化。结果实验组和阳性对照组BALB/c小鼠肺组织呈现明显的炎症性病理改变;rDerf2组的BALF中白细胞总数〔(17.39±1.03)×106/L〕及嗜酸性粒细胞(EOS)〔(1.61±0.03)×106/L〕计数,均明显高于PBS组(P<0.01);BALF中IL-4〔(78.92±9.06)pg/ml〕、IL-17〔(201.63±31.26)pg/ml〕与PBS组比较呈明显的高水平表达(P<0.01);而IL-2〔(8.29±1.27)pg/ml〕和IFN-γ〔(51.04±15.85)pg/ml〕的含量则显著下降(P<0.01);上述指标在脾细胞培养上清中出现类似的变化。rDerf2组血清中IgE〔(37.63±6.57)IU/ml〕、IgG1〔(16.68±2.90)μg/ml〕的含量表现为Th2型反应增强趋势;但OVA组和rDerf2组间所有指标差异均无统计学意义(P>0.05)。结论用rDerf2腹腔注射致敏后再雾化激发的方式能够成功构建小鼠肺部变应性炎症模型。     

小鼠过敏性肠炎模型的免疫学研究

目的 评价卵清蛋白(OVA)饲喂致敏OVA特异性T细胞受体转基因小鼠(OVA23-3)的方法建立过敏性肠炎模型的可行性,并观察其免疫学指标变化.方法 采用高OVA饲料饲喂OVA23-3小鼠的方法,建立小鼠过敏性肠炎模型.通过ELISA法检测血清OVA特异性IgE抗体和细胞培养上清细胞因子的含量,流式细胞术检测细胞内细胞因子.结果 高OVA饲料饲喂引起OVA23-3小鼠体重下降,并发生以小肠为主的出血性炎症.与对照组比较,实验组血清OVA特异性IgE含量明显增高,细胞培养上清IL-4水平增高,而IFN-β水平降低,同时CD4+T细胞中IL-4产生细胞百分率显著增加,IFN-γ产生细胞百分率显著减少.结论 高OVA饲料饲喂OVA23-3小鼠诱发过敏性肠炎的方法是一种理想快速简便的方法,Th2型细胞反应占优势是其重要特征.     

内毒素在小鼠过敏性哮喘模型所起作用的实验研究

目的建立小鼠过敏性哮喘模型,在过敏原的致敏和激发阶段给予不同剂量内毒素(即脂多糖LPS),探讨内毒素在哮喘模型中的作用机制。方法建立BALB/c小鼠哮喘模型,设立实验组和对照组开展后续实验。实验组包括:鸡卵清白蛋白(OVA)致敏,OVA激发组;OVA致敏,LPS激发组;OVA致敏,OVA+LPS联合激发组;LPS暴露于OVA致敏前,OVA激发组;对照组包括试剂对照组和健康对照组。各实验组的LPS剂量均分为高、中、低三个剂量组。通过测定小鼠气道阻力和肺顺应性来反映其肺功能改变;流式细胞术(FCM)测定FITC、PE阳性细胞数,表征CD4+CD2+5调节性T淋巴细胞(以下简称Treg细胞)的相对比例;荧光实时聚合酶链反应定量测定肺脏和脾脏Foxp3mRNA表达的水平;ELISA法测定血浆总IgE和OVA特异性IgE水平,抗体夹心ELISA法测定支气管肺泡灌洗液(BALF)中Th2细胞因子(IL-4和IL-5)的水平;计数BALF中白细胞总数和嗜酸性粒细胞等各类白细胞所占百分比;常规病理切片观察肺组织改变情况。结果结果表明,与哮喘模型组小鼠相比,在LPS各处理组中,总体上能诱导Treg细胞的产生,改善肺功能,降低IgE滴度,降低Th2介导的免疫反应,同时诱导肺部炎症。尤其是在致敏阶段腹腔注射内毒素可以缓解由过敏原诱导的气道炎症和改善肺功能,诱导肺组织和脾组织Foxp3mRNA表达的增加。结论LPS抑制了Th2介导的过敏反应,可能与Foxp3表达量的增加相关,后者诱导Treg细胞增殖,对哮喘症状起到缓解作用。     

Gastric Enzyme Supplementation Inhibits Food Allergy in a BALB/c Mouse Model

Impaired gastric digestion due to suppressed gastric acidity enhances the risk for food allergy development. In the current study, we aimed to evaluate the impact of a supported gastric digestion via application of a pharmaceutical gastric enzyme solution (GES) on food allergy development and allergic reactions in a BALB/c mouse model. The ability of the GES to restore hypoacidic conditions was tested in mice treated with gastric acid suppression medication. To evaluate the impact on allergic symptoms, mice were orally sensitized with ovalbumin (OVA) under gastric acid suppression and subjected to oral challenges with or without GES. The immune response was evaluated by measurement of antibody titers, cytokine levels, mucosal allergy effector cell influx and regulatory T-cell counts. Clinical response was objectified by core body temperature measurements after oral OVA challenge. Supplementation of GES transiently restored physiological pH levels in the stomach after pharmaceutical gastric acid suppression. During oral sensitization, supplementation of gastric enzymes significantly reduced systemic IgE, IgG1 and IgG2a levels and allergic symptoms. In food allergic mice, clinical symptoms were reduced by co-administration of the gastric enzyme solution. Support of gastric digestion efficiently prevents food allergy induction and alleviates clinical symptoms in our food allergy model.     

Food Allergen Nitration Enhances Safety and Efficacy of Oral Immunotherapy in Food Allergy

(1) Background: Posttranslational protein modifications have been demonstrated to change protein allergenicity. Previously, it was reported that pretreatment with highly nitrated food proteins induced a tolerogenic immune response in an experimental mouse model and in human immune cells. Here, we investigated a possible therapeutic effect of modified proteins and evaluated the safety of oral exposure to highly nitrated proteins in an experimental food allergy model. (2) Methods: BALB/c mice were orally sensitized towards ovalbumin (OVA) under gastric acid suppression. Thereafter, treatment via intragastric gavage with maximally nitrated OVA (nOVAmax) and OVA as a control was performed six times every 2 weeks. On the last day of experiments, all the treated mice were orally challenged with OVA. Systemic anaphylactic reaction was determined by measuring the core body temperature. Moreover, antibody levels, regulatory T cell numbers, cytokine levels and histology of antrum tissues were analyzed. (3) Results: After oral immunotherapy, OVA-specific IgE titers were decreased while IgG1 titers were significantly elevated in the mice receiving OVA. After oral challenge with OVA, nOVAmax-treated allergic animals showed no drop of the core body temperature, which was observed for OVA-allergic and OVA-treated allergic animals. Significantly fewer eosinophils and mast cells were found in the gastric mucosa of the allergic mice after nOVAmax treatment. (4) Conclusions: Oral immunotherapy with nOVAmax reduced allergic reactions upon allergen exposure and the number of allergen effector cells in the gastric mucosa. Thus, maximally nitrated allergens enabled an efficient and safe treatment for food allergy in our experimental model.     

槐耳颗粒对哮喘模型TGF-β表达的干预作用

目的:观察槐杞黄颗粒对哮喘模型肺组织转化生长因子-β(transforming growth factor-β,TGF-β)表达的干预作用。方法:将60只BALB/c哮喘模型鼠随机分为空白对照组、哮喘模型组及槐杞黄颗粒干预组(每组20只);除空白对照组外其他各组小鼠在试验第1天、第7天和第14天通过腹腔注射0.2 ml混有40 mg氢氧化铝和10μg OVA的pH7.4的PBS液致敏,在试验第15天用5%的OVA对小鼠进行雾化激发,连续一周;槐杞黄颗粒干预组在致敏阶段即每日加用槐杞黄颗粒胃内注入5 g/d,时间为1个月;所有小鼠在实验1个月后杀死,分别用ELISA以及免疫组化方法检测血中IgE、IL-4水平以及肺组织TGF-β的表达。结果:槐杞黄颗粒干预组1个月后血中IgE、IL-4水平及肺组织TGF-β的表达明显低于哮喘模型组,血中的IFN-γ水平较哮喘模型组明显增多,差异均有统计学意义(P<0.05)。结论:槐杞黄颗粒长期使用可能会逆转过敏性炎症,并且对哮喘模型肺组织TGF-β的表达有明显的抑制作用。     

过敏性鼻炎特异性IgE的构成及影响因素分析

目的分析过敏性鼻炎特异性IgW的构成及相关的影响因素,为过敏性鼻炎的预防提供数据支持。方法根据患者的人口学变量、环境暴露因素、家族过敏遗传史、生活习惯等内容设计调查问卷,对疑似有过敏症状的患者进行问卷调查,结合调查问卷与实验室检测结果,分析过敏性鼻炎特异性IgE的构成。采用多因素logistic回归,分析过敏性鼻炎的影响因素。结果年龄段在18—39岁、居住在城市以及具有本科或本科以上学历的人群,过敏性鼻炎患病率最高（18．69％,19．00％,22．62％）。矮豚草蒿过敏原是过敏性鼻炎特异性IgE构成中构成比最大、IgE浓度最高的过敏原（构成比36．30％,几何均值7．4IU／m1）。在控制年龄、生活地区和文化程度因素后,过敏性鼻炎的危险因素有：母亲过敏史、食物过敏史和矮豚草蒿过敏。结论预防过敏性鼻炎的重点人群包括：母亲有过敏史和食物过敏。对矮豚草蒿过敏的人群应及时进行脱敏治疗从而降低过敏性鼻炎的发病风险。     

Class specific influence of dietary Spirulina platensis on antibody production in mice

In the present study, we investigated antibody productions of IgA and other classes, such as IgE and IgG1, in mice as possible evidence of the protective effects of Spirulina toward food allergy and microbial infection. An increase of IgE antibody level in the serum was observed in the mice that were orally immunized with crude shrimp extract as an antigen (Ag group). The antibody level, however, was not further enhanced by treatment with Spirulina extract (SpHW). IgG1 antibody, on the other hand, which was increased by antigen administration, was further enhanced by Spirulina extract. It was noted that the IgA antibody level in the intestinal contents was significantly enhanced by treatment with Spirulina extract concurrently ingested with shrimp antigen, in comparison with that of the Ag group treated with shrimp antigen alone. An enhancement of IgA antibody production by Spirulina extract was also observed in culture supernatant of lymphoid cells, especially in the spleen and mesenteric lymph node from mice treated with Spirulina extract for 4 weeks before antigen stimulation. These results suggest that Spirulina may at least neither induce nor enhance allergic reaction such as food allergy dependent on an IgE antibody, and that when ingested both concurrently with antigen and before antigen stimulation, it may significantly enhance the IgA antibody level to protect against allergic reaction.     

Nonmurine animal models of food allergy

Food allergy can present as immediate hypersensitivity [manifestations mediated by immunoglobulin (Ig)E], delayed-type hypersensitivity (reactions associated with specific T lymphocytes), and inflammatory reactions caused by immune complexes. For reasons of ethics and efficacy, investigations in humans to determine sensitization and allergic responses of IgE production to innocuous food proteins are not feasible. Therefore, animal models are used a) to bypass the innate tendency to develop tolerance to food proteins and induce specific IgE antibody of sufficient avidity/affinity to cause sensitization and upon reexposure to induce an allergic response, b) to predict allergenicity of novel proteins using characteristics of known food allergens, and c) to treat food allergy by using immunotherapeutic strategies to alleviate life-threatening reactions. The predominant hypothesis for IgE-mediated food allergy is that there is an adverse reaction to exogenous food proteins or food protein fragments, which escape lumen hydrolysis, and in a polarized helper T cell subset 2 (Th2) environment, immunoglobulin class switching to allergen-specific IgE is generated in the immune system of the gastrointestinal-associated lymphoid tissues. Traditionally, the immunologic characterization and toxicologic studies of small laboratory animals have provided the basis for development of animal models of food allergy; however, the natural allergic response in large animals, which closely mimic allergic diseases in humans, can also be useful as models for investigations involving food allergy.     

Hypersensitivity linked to exposure of broad bean protein(s) in allergic patients and BALB/c mice

ObjectiveBroad bean (Vicia faba L.), a common vegetable, belongs to the family Fabaceae and is consumed worldwide. Limited studies have been done on allergenicity of broad beans. The aim of this study was to determine if broad bean proteins have the ability to elicit allergic responses due to the presence of clinically relevant allergenic proteins.MethodsSimulated gastric fluid (SGF) assay and immunoglobulin E (IgE) immunoblotting were carried out to identify pepsin-resistant and IgE-binding proteins. The allergenicity of broad beans was assessed in allergic patients, BALB/c mice, splenocytes, and RBL-2H3 cells.ResultsEight broad bean proteins of approximate molecular weight 70, 60, 48, 32, 23, 19, 15, and 10 kDa that remained undigested in SGF, showed IgE-binding capacity as well. Of 127 allergic patients studied, broad bean allergy was evident in 16 (12%). Mice sensitized with broad bean showed increased levels of histamine, total and specific IgE, and severe signs of systemic anaphylaxis compared with controls. Enhanced levels of histamine, prostaglandin D₂, cysteinyl leukotriene, and β-hexosaminidase release were observed in the primed RBL-2H3 cells following broad bean exposure. The levels of interleukin IL-4, IL-5, IL-13 and regulated on activation, normal T-cell expressed and secreted were found enhanced in broad bean-treated splenocytes culture supernatant compared with controls.ConclusionThis study inferred that broad bean proteins have the ability to elicit allergic responses due to the presence of clinically relevant allergenic proteins.     

IgE介导食物过敏和婴幼儿特应性皮炎的相关性研究

目的 探讨婴幼儿IgE介导的食物过敏和特应性皮炎(AD)的相关性.方法 选取2014年1月至2016年12月在济宁市第一人民医院皮肤科和儿科变态反应门诊确诊为AD的婴幼儿80例,年龄3个月至3岁.详细询问病史并填写食物过敏调查问卷,再行血清食物特异性IgE(sIgE)检测.除极高水平sIgE外,sIgE阳性患儿需要口服食物激发试验(OFCs),采用二元Logistic回归分析食物sIgE阳性分级对诊断食物过敏的影响.结果 26(32.50%)例AD患儿有食物过敏史或可疑食物过敏史.食物sIgE阳性38例,占总数的47.50%.不同程度的AD患儿在一种食物sIgE阳性和两种及以上食物sIgE阳性比较差异有统计学意义(χ2=12.88,P<0.05).其中极高水平sIgE(6级)5例.结合过敏史、sIgE阳性和OFCs,确诊食物过敏18例(鸡蛋12例、牛奶5例、鱼1例),总阳性率为22.50%.食物sIgE阳性分级越高,IgE介导的食物过敏发生率越高(Waldχ2=13.24,P<0.05,OR=2.81).结论 婴幼儿AD与IgE介导的食物过敏存在密切关联性.个人史和家族过敏史为诊断提供线索,sIgE分级为诊断提供有价值的参考,确诊需要OFCs.牛奶和鸡蛋是婴幼儿最常见的过敏原.