Study On The Differentiation Of Secreting IgE In Tonsil Of 10 Different Allergen Induced Allergic Rhinitis

Objective:Allergic rhinitis?AR?refers to the release of inflammatory mediators mediated by IgE?immunoglobulin E,IgE?with susceptible individuals after exposure to allergens,and is accompanied by a variety of immune active cells and cytokines,such as^[1-4],a chronic inflammatory response disease of the nasal mucosa.The in-depth understanding of the pathogenesis of allergic rhinitis is of great significance for preventing and thoroughly eradicating this disease.According to the literature,the palatal tonsils,which are formed after culture in vitro,will differentiate IgE+B lymphocytes^[5].But in the palatine tonsil of allergic rhinitis,will there still be the same highly differentiated expression of IgE+B lymphocytes in vitro?What is the difference in the expression of IgE+B lymphocytes in the palatine tonsil of non allergic rhinitis patients?None of them have been confirmed.In the palatine tonsils of allergic rhinitis,the various subtypes of B lymphocytes:plasma cells,germinal center B cells,memory B cells,and naive B cells will be changed more than normal subjects,and how they relate to IgE+B lymphocytes will be an important direction of our experimental analysis.Research methods:1.Sample sourceFrom June 2016 to September 2017 in our hospital department of ENT the implementation of tonsillectomy and adenoidectomy and clinical diagnosis of allergic rhinitis and inhalant allergens of at least one positive patients with tonsil and?or?adenoid samples,a total of 26 cases,there were 16 males,10 females,age 6 to 51 years old,the average age was 26.27 years old,and the exclusion of other autoimmune diseases,blood diseases,tumor,liver and kidney disease and acute and chronic infection disease.At the same time,15 tonsil tissues of all patients with negative allergic rhinitis and inhaled allergens were selected,including 11 males and 4 females,aged from 6 to 54 years old,with an average age of 26.13 years.2.ReagentsAllergen specific IgE antibody test paper?Suzhou Hao OBO bio Pharmaceutical Co.,Ltd.?Enzyme labeled antibody binding solution?Suzhou Hao o o Bo bio Pharmaceutical Co.,Ltd.?Bovine alkaline phosphatase?ALP?labeled rat anti human IgE antibody solution?Suzhou Hao o o Bo bio Pharmaceutical Co.,Ltd.?FITC labeled mouse anti human IgE polyclonal antibody?BD Pharmingen?PerCP labeled mouse anti human CD19 polyclonal antibody?BD Pharmingen?PE labeled mouse anti human CD27 polyclonal antibody?BD Pharmingen?FITC labeled mouse anti human CD38 polyclonal antibodies are purchased from BD Pharmingen in the United States.Human peripheral blood lymphocyte separation medium?Tianjin City Hao Yang Biotechnology Co.,Ltd.?PBS fluid?laboratory self matching?D-Hank's?laboratory self matching?RPMI1640?laboratory self matching?3.Clinical data collectionThe venous blood of 5ml patients was collected and used as an allergen specific IgE antibody test.The patient's medical records were collected and the relevant data were collected.The diagnosis of the disease was based on the Lanzhou standard of the Chinese Otorhinolaryngology Head and neck surgery.4.Detection ogE antibody in venous blood allergensFor each patf specific Iient,3 reacting tubes were prepared.Take the sample into the position of the first translation officer's projection line,put the test bar into the test bar,incubate at room temperature for 6 hours or overnight.Take out the test strip and rinse for 55 seconds at the bottom of the water flow.The flow of water must have a certain impulse and keep it constant.Move the test strip back and forth in the flow of water to ensure that the detection area is fully cleaned.In the second reaction tubes,add the enzyme labeled antibody binding liquid to the position of the marking line,then flush the test strip into the reaction tube and incubate for 30 minutes at room temperature.Take out the test strip and wash it at the bottom of the water stream until the red is completely disappearing.Move the test strip back and forth in the flow of water to ensure that the detection area is fully cleaned.In the third reaction tubes,the substrate liquid is added to the position of the scale line of the protuberance,and the flushed test strip is reacted to Guan Zhong and incubated for 30 minutes at room temperature.Remove the test strip from the reaction tube and dip it in the water absorbent paper.Dry at room temperature for at least 30 minutes and read the results.5.Flow cytometry antibody stainingTonsil and adenoid samples after washing after cutting into grinding homogenate,suspending centrifugal washing after density gradient centrifugation to extract mononuclear cells,mononuclear cells isolated from the adjusted cell number after placed in a centrifuge tube,take 100ul into cell suspension flow tube,joined the IgE,the 1ul CD19 antibody.And after mixing,flow tube into the tube holder with aluminum foil wrapped into the reaction of 30min 4^C refrigerator.In the other first class tube,CD27,CD38 antibody was used for surface staining.The expression of IgE and the subtype of B cell were determined.6.Data processingSPSS 20 statistical software was used to analyze the data.The detection data were expressed as x+s,two independent samples were compared with paired sample t test,and two variable correlation analysis was analyzed by Spearman correlation analysis.The difference between P<0.05 was statistically significant.Result:1.Allergen results of inhalation of 10 allergensIn 26 patients with allergic rhinitis,after 10 allergen specific IgE antibodies,the positive rate of allergens was from high to low:15 cases of dust mites,10 cat hair,10 dog epithelium,6 cases of Artemisia,5 moulds,4 cases,4 cases,and the same patient with the most positive reaction to 4 allergens.It is suggested that desensitization therapy for a single allergen may not be effective.2.Differential expression of IgE⁺B lymphocytes in palatine tonsil of allergic rhinitis group and non allergic rhinitis groupIn the allergic rhinitis group,we found that the differential expression of IgE⁺B lymphocytes was significantly higher than that in the non allergic rhinitis group.3.The relationship between B lymphocyte subtypes and differential expression of IgE⁺B lymphocytes in palatine tonsil of allergic rhinitis groupIn the allergic rhinitis group,the percentage of differential expression of B cells and IgE+B lymphocytes in the germinal center increased significantly.4.The differential expression of B cells and memory B cells and IgE⁺B lymphocytes in the germinal center of allergic rhinitis groupThe differential expression of IgE⁺B lymphocytes was positively correlated with the percentage of B cells in the germinal center,and negatively correlated with B cells.Conclusion:1.The differential expression of IgE⁺B lymphocyte in the palatine tonsil of the allergic rhinitis caused by 10 allergen groups was higher than that of the non allergic rhinitis,which was consistent with the increase of the specific IgE in the blood of allergic rhinitis.2.In the allergic rhinitis group,the percentage of B cells and plasma cells in the germinal center increased.It reflects the transformation ability of B lymphocytes from germinal center to IgE+B lymphocytes.At the same time,plasma cells as effector cells secreting IgE increase the expression of IgE⁺B lymphocytes at the same time as plasma cells increase.3.in the B lymphocyte subsets of the allergic rhinitis,the percentage of B cells in the germinal center was positively correlated with the differential expression of specific IgE,while the percentage of the memory B cells was negatively correlated with the differential expression of specific IgE,suggesting that the germinal center B cells in the allergic rhinitis would increase the IgE⁺B lymphocyte,and the memory B cells would make IgE⁺B lymphocyte decrease.