| The intestinal sensitization is the key factor of food allergy because the intestinal mucosal immunity reaction in gastrointestine could be caused by the antidigestion of some food allergen proteins and/or by the more exposure of antigen epitopes on some protein digested products.When food is digested in the gastrointestinal tract,proteins will be gradually degraded to form large molecular fragments,small molecular peptides and amino acids,which will be absorbed and utilized by the human body in the intestinal tract.During this process,the allergen protein will undergo structural changes under the influence of gastrointestinal digestive enzymes and gastric acid,thus destroying the original antigen epitopes or exposing the hidden antigen epitope.After the digestive fragment including the exposed epitope are transported in the intestine,allergic reaction is caused in the body.Eggs are the second most common food allergen.Ovotranferrin as the main egg allergen which exists in egg white,is said be similar to ovomucinoid.At present,the researches about ovotransferrin mainly focus on processing.The previous studies our research group did suggested that ovotransferrin digestive products maybe still had strong potential sensitization.Based on this,this paper continue to carry out an evaluation experiment on the sensitization of the simulated gastrointestinal digestive products of ovotransferrin and expect to further understand the role of ovotransferrin's simulated digestive products in the important stage of food allergy.In this study,ovotransferrin from egg white as a subject,it used to simulate digestion of gastric juice,small intestinal juice and small intestinal brush membrane enzyme in vitro,to evaluate the ability of simulated infant and adult digestive products fragments to bind to specific antibodies.The Caco-2 cells monolayer was used to determine the transport capacity of ovotransferrin's simulated digestive products on intestinal epithelial cells,studied the effects of them on tight junction proteins of intestinal epithelial cells.Mouse bone marrow dendritic cells were used to detect the uptake rates of ovotransferrin's simulated digestion products,and to evaluate their immune-stimulating ability.BALB/c mice were used to construct a sensitization model.According to the clinical symptoms and histomathology of the sensitized mice,as well as the content of specific antibodies in the sera,cytokines secretion in spleen cells and T cell subsets differentiation,the sensitization of ovotransferrin's simulated digestive products were evaluated.At the same time,combined with the expression of dendritic cells and different T cell subsets in the mesenteric lymph nodes of the intestinal mucosal immune system,to evaluate the effects of ovotransferrin's simulated digestive products on the intestinal mucosal immune system,and further illustrated the sensitization mechanism of ovotransferrin's simulated digestive products through the gastrointestinal digestion in vitro.The main research methods,results and conclusions are as follows:(1)Ovotransferrin from egg white was extracted by weak cation exchange chromatography combined with hydroxyapatite chromatography.After SDS-PAGE,the purity of ovotransferrin was above 95%.After linear amplification of the purification system,gram level ovotransferrin could be obtained after one purification cycle without any change in purity.Tricine-SDS-PAGE was used to characterize the simulated products of ovotransferrin digested by simulated infant and adult digestive systems in vitro,and it was found that part of ovotransferrin was still undigested in the simulated infant digestive products.There was almost no undigested ovotransferrin in the simulated adult digestive products,and the larger molecular weight fragments in the products were also less than those of simulated infant digestive products.Followed by western-blotting to evaluate the potential allergenicity of ovotransferrin's simulated digestive products.The results showed that both of the two simulated digestive products have the fragments which can combine with specific Ig G antibody and has potential allergenicity to some extent.The simulated infant digestive products can be combined with fragments more,potentially allergenicity also relatively stronger than simulated adult digestive products.(2)A Caco-2 cells monolayer model which has well integrity was established in the experiment.It was found that ovotransferrin's simulated digestive products could transport on the Caco-2 cells monolayer.Within the same transport period,the highest transport rate was ovotransferrin,followed by simulated infant digestive products.Immunofluorescence experiments about the damage of tight junction proteins Occludin and ZO-1 were conducted on the the Caco-2 cells monolayer after transport test.Compare to simulated adult digestive products,simulated infant digestive products cause the more serious damage.In addition,we extracted bone marrow dendritic cells which the purity reached 88%.The bone marrow dendritic cells were co-incubated with fluorescently labeled ovotransferrin's digestive products.Flow cytometry was used to detect various fluorescence expressions.The results showed the lowest taken rate was ovotransferrin,the taken rate of simulated infant digestive products higher than that of simulated adult digestive products.However,ovotransferrin's digestive products had no significant effect on the expression of costimulatory molecules CD40,CD86 on the surface of bone marrow dendritic cells,and only the simulated infant digestive products up-regulated the expression of functional factor CD103 on the surface of dendritic cells.(3)An oral sensitization animal model of ovotransferrin's digestive products was established to evaluate the sensitization of them.In terms of anaphylactic symptoms,changes in body temperature,pathological changes of jejunum and lung tissues,the ovotransferrin group had the worst results.The symptoms of simulated digestive products group were mild.We test the levels of specific antibodies related to Th2 in the sera including Ig E,Ig G and Ig G1 by indirect ELISA assay.Compared with simulated adult digestive products,simulated infant digestive products significantly upregulated antibodies levels.MSD kits were used to detect various cytokines secreted by spleen cells,and the results showed that simulated infant digestive products could stimulate higher secretion of Th2 cytokines and lower secretion of Th1 cytokines by spleen cells than simulated adult digestive products.Different subsets of T cells in spleen cells were labeled and detected by flow cytometry.The results showed that simulated infant digestive products could increase the expression of Th2 cells and decrease the expression of Th1 cells more significantly than simulated adult digestive products.These results suggested that ovotransferrin and its digestive products could promote the shift of allergic reaction towards Th2 and had certain allergenicity,the allergenicity of ovotransferrin is the highest,followed by simulated infant digestive products,simulated adult digestive products is the lowest.(4)The effect of ovotransferrin on intestinal mucosal immune system was investigated by mouse sensitization model.The expression of various cells in mesenteric lymph nodes was analyzed by flow cytometry.The expression of CD11c+CD103+ dendritic cells and CD4+T cells in MLN of intestinal mucosal immune system of mice were not significantly affected by ovotransferrin and its simulated digestion products,but the expression of CD8+T cells was significantly down-regulated by ovotransferrin.However,both ovotransferrin and its simulated digestive products could decrease the expression of Th1 cells and increase the expression of Th2 cells in MLN significantly.Compared with control group,the simulated adult digestive products upregulated the expression of Th2 cells in MLN(P < 0.001)and down-regulated the expression of Th1 cells(P < 0.001)to less extent.These data showed ovotransferrin significantly disrupt the balance of intestinal mucosal immune system,followed by simulated infant digestive products. |
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