| OBJECTIVE:Allergic rhinitis is an allergic disease which occurred in nasal mucosa, it mainly attack on children and young adults. According to the WHO data recently, there are more than 500 million people worldwide suffering from the disease currently. Epidemiological investigation showed that in the United States, the incidence of allergic rhinitis is as high as 16.7%in 2008, which ranks fifth in the chronic disease, and is one of the most common chronic disease in children, the expense of AR is billions of dollars each year. In our country, epidemiological investigation data from 11 central city in 2007, adult self-reported prevalence was between 9-24.6%. In recent 10 years, the reported incidence of allergic rhinitis in children also have differences in domestic data, it is between 1.8%to 13.67%. Allergic rhinitis itself is not a serious disease, but can significantly affect the quality of life and health. At present, the most common protocol that treat allergic rhinitis is intranasal corticosteroids and oral antihistamines, although this method can quickly control the allergic inflammation and relieve the clinical symptoms, but it can’t prevent allergic rhinitis break out repeatedly, so we are still far from cure target. Study shows that only about 60% of the AR patients feel very satisfied with the treatment, and as time goes on, the efficacy of drug treatment will gradually weaken, these difficulties in treatment lies in the pathogenesis of allergic rhinitis not being fully understood, so it is difficult to explore more effective treatments. Therefore, the study of pathogenesis and prevention measures is becoming the highlights in the otolaryngology field.As we all known, the pathogenesis of AR is very complex, it is mainly related to atopic individuals, genetic background and environmental factors. A large amount of experimental study confirmed that the pathogenesis of nasal allergy give priority toTh2 reaction, namely the downstream biological effects caused by Th1/Th2 imbalance lead to the occurrence of AR, but recently some scholars put forward new subgroup of T cell, such as regulatory T cells may regulate Th2 cells differentiation, CD4+CD25+Treg cells play a strong inhibitory effect on Th2-mediated respiratory allergic inflammation. Atopic individuals may include Treg/Th2 imbalance, but its complex signal transduction pathways and mechanisms are still in the exploration, which will provide new therapeutic targets for allergic rhinitis.DCs are the most powerful professional antigen presenting cells in the body, and are also the unique APC which can make the initial T cell activation, they are the central part of the immune response at immunologic priming, regulation, and maintain. Studies have confirmed that Th2 polarization in the allergic rhinitis is associated with the dysfunction of DC cells, DC is becoming the important target cells which influence T cell activation and proliferation. Because of lacking the surface stimulating factors, Immature DCs showed their characteristics of inducing Treg cells in vivo or in vitro, therefore we adopt genetic modification method to decorate DC, in order to keep the stability of the immature DC, thus suppress effective T cell activation and induce Treg cells differentiation.This study intends to take AR as the research object, to clarify whether there is Treg/Th2 imbalance or not in AR, we also intends to take DC-Th interaction as the main axis, and use lentiviral vector CD86-siRNA transfect and regulate DC cells, to elaborate the upstream events of Treg/Th2 imbalance and immune regulatory factors, the purpose is to provide new strategies for the pathogenesis of AR.METHODS:1. Grouping of study objects and specimen collection:strictly according to the guidance of diagnosis and treatment for allergic rhinitis from Chinese medical association (2009, wuyi mountain), all patients were from the children hospital of Chongqing medical university and were diagnosed for the first time, and they did not receive relevant treatment for AR, the control patients exclude allergic diseases, after finishing informed consent, they were bringed into the study.2. We explore Treg/Th2 cells subsets in peripheral blood of AR patients and controls and its relationship with clinical symptoms, the expression of Treg/Th2 cytokines and transcription factor.(1) We detected the levels of IgE, IL-4, IL-5, TGF-β1 in peripheral blood of AR patients and controls by ELISA kits.(2) By RT-PCR, we detected the expression of FOXP3, GATA-3 transcription factors.(3) By Western Blot, the levels of FOXP3, GATA-3 protein in the peripheral blood of AR patients and normal controls being measured.3. lentiviral vector CD86-siRNA sequences transfect dendritic cells to silence CD86 genes, through this method, we observed its influence on Treg/Th2 cells differentiation.(1) construction of lentiviral vector CD86-siRNA(2) Sorting CD14+monocytes from PBMC by MACS and inducing mature DC differentiation(3) Sorting I CD4+T cells from PBMC by MACS(4) lentiviral vector CD86-siRNA sequences transfect dendritic cells(5) Co-culture of untransfected DC, transfected DC,normal DC with CD4+T cells from peripheral blood, We detected the levels of IL-4, IL-5, TGF-β1 in supernatant by ELISA kits, the expression of FOXP3, GATA-3 transcription factors by RT-PCR and the levels of FOXP3, GATA-3 protein through Western Blot, therefore we can evaluate the effects on Th cell differentiation through modified DC which were silenced CD86 gene.RESULTS:(1) In AR group, compared with controls, serum total IgE level, eosinophil ratio, T5SS were significantly increased, serum total IgE level and eosinophil ratio were positively correlated with T5SS, but TGF-β1/ IL-4, TGF-β1/IL-5,FOXP3mRNA/GATA3 mRNA, FOXP3 protein/GATA3 were negatively correlated with T5SS.By ELISA kits, AR group compared with control group, the levels of IL-4, IL-5 were significantly increased, but the expression of TGF-β1 was obviously lower than control group.The expressions of FOXP3 mRNA and GATA3 mRNA were detected by RT-PCR, in AR group, FOXP3 mRNA was significantly lower, but GATA3 mRNA was clearly higher than control group. FOXP3 and GATA3 proteins were measured through Western blotting, the results kept accordance with their mRNA levels(2) We successfully extracted CD14+ monocytes from PBMC by MACS and stimulus them into mature DC, we also successfully extract the CD4+ T lymphocyte from PBMC by MACS, and establish co-culture system (CD14+/CD4+T1:5); Using lentiviral vector CD86-siRNA sequences transfect dendritic cells at MOI 20, the transfection rate was about 60.2%; Co-culture of untransfected DC in AR, transfected DC in AR, normal DC with CD4+T cells from peripheral blood, We detected the levels of IL-4, IL-5, TGF-β1 in supernatant. After transfection, Th2 type cytokines IL-4, IL-5 were lower than that in untransfected group, but still significantly higher than that of normal control group, Treg cytokine TGF-β1 in untransfected AR group is lower than normal controls, after siRNA transfection it is significantly higher than the untransfected group, but they were still lower than the control group; Th2 type transcription factor GATA3 mRNA and protein keep accordance with Th2 type cytokines IL-4, IL-5, and Treg type transcription factor FOXP3 mRNA and protein appear no difference with TGF-β1.CONCLUSIONS:(1) In AR patients, FOXP3 mRNA and protein were significantly lower compared with subjects in control. But GATA3 mRNA and protein were higher in AR. FOXP3 mRNA/GATA3 mRNA, FOXP3 protein/ GATA3 protein were negatively correlated with T5SS, this study illustrate another immune imbalance(Treg/Th2 imbalance) may exist in AR.(2) In our study, lentiviral vector with CD86-siRNA sequences were transfected into human DCs to make CD86 gene silence. As a result, the surface molecule CD86 of decreased dramatically. Then Treg/Th2 cells differentiation were influenced by co-culture experiments of DCs/CD4+T cells in vitro. So we reveal that T cell differentiation can be regulated by genetic modification of DC, which change the balance of Treg/Th2. From the study, we confirm that DC-Th axis may play an important role in the mechanism of AR. This study may provide a new idea for the treatment of AR. |
PDF Full Text Request