| Allergic rhinitis(AR) refers to a chronic inflammatory disease which happens to the nasal mucosa after the atopic individuals exposed to allergen. This process is mediated by IgE, with a variety of immune cells and cytokines involved. The patients with AR are often manifested as nasal itching, sneezing, urbid nasal discharge, eye itching and other symptoms. With people’s living standards improving, the incidence of AR are increasing year after year. The cost for the treatment of AR is so expensive that it brings a heavy burden to both the family and the society. At present, the therapeutic methods to AR include desensitization therapy and symptomatic treatment, which are not very effective. That is because the easy relapse and poor patient compliance. AR is often complicated by the asthma, which has a serious impact on the quality of the patients’ lives. Therefore, to find an effective method to prevent and cure AR is of a great significance. Recently, studies have shown that the incidence of allergic diseases was significantly higher in the less developed countries that the developed ones. While the reason for this cannot be explained alone by genetic factors such as racial differences. Many scholars believed that the decrease of the microbial diversity in the living environment could be related to some immune and chronic inflammatory diseases, such as Atopic dermatitis and Crohn’s disease. Strachan’s "hygiene hypothesis" proposed that with the improving of the human living environment and more and more attention to the surrounding health conditions, the chance for human especially children to contact with pathogenic microorganisms reduces significantly. The result of the reducing chances is that the children’s immune system lack the effective stimulation and mobilization, and it triggers the excessive reaction of the immune system to the micro flora that should belong to the normal ones. At last, it leads to the increased incidence of allergic diseases. Then does the occurrence of AR have relevance to the changes of nasal micro ecological environment? Does the nasal micro ecological environment change in patients with AR? In order to answer the questions above, we designed and carried out the experiments to find out the changes of the nasal microbiogeocoenosis on AR patients and the effect and mechanism of the changes on AR. There are three parts in our study. Study Ⅰ This study was designed to find out the impact of micro-ecological environment on the incidence of allergic rhinitis after developing a model of allergic rhinitis on mice.Method: 60 mice were randomly divided into GF group(n=30) and SPF group(n=30). Mice of GF group were fed in the germ-free environment and mice of SPF group were fed in the specific pathogen-free environment. Then each group were randomly divided into Model group(20 mice) and Control group(10 mice).Establish allergic rhinitis model in the mice of model group using ovalbumin(OVA) at the age of 6 weeks, observe and score the corresponding symptoms and signs that could been induced. Stain with hematoxylin eosin(HE) staining method for nasal mucosa to observe the morphological changes. Using enzyme linked immunosorbent assay to detect the concentration of IgE, IFN- γ and IL-4 in the peripheral blood serum.Results: The chi square test shows that the incidence of allergic rhinitis in the mice of GF group is significantly higher than that of the SPF group(P<0.05).HE staining shows that the nasal mucosas of allergic rhinitis positive reaction mice are highly hyperemia and edema and have a large number of inflammatory cell infiltration, while there is no abnormal morphology of nasal mucosas in mice with no allergic rhinitis reaction. EOS counting display that the number of eosinophilic cells in nasal mucosa of positive allergic rhinitis reaction mice is increased significantly. The concentration of IgE and IL-4 in the serum of positive allergic rhinitis reaction mice is highly increased(P<0.05), and IFN-γ is significantly decreased(P<0.05).Conclusion: The incidence of allergic rhinitis in the mice of GF group is significantly higher than that of the SPF group. The difference of micro-ecological environment may play a key role in the occurrence of allergic rhinitis in mice. Study Ⅱ The second study is to explore the difference of micro environment flora in the nasal cavity of AR patients and normal persons by 16 S rDNA sequence analysis.Method: Take the nasal secretions of 8 AR patients and 7 normal persons with a sterile swab under nasal endoscope. Then extract the total bacterial DNA of each sample, and detect for the 16 S rDNA V6 region.Results: The quantity of data produced was 1900.09 Mb after sequenced. The range of total bacteria detection covered 35 phylum, 39 classes, 77 orders, 179 families, 338 genera and 38 species. With analysis and comparison, the category of bacteria in the nasal cavity of AR patients(3176±623.6) was higher than that of normal persons(1771±397.2). Principal component analysis(PCA) showed that the differences of Actinomycetales, Lactobacillales and Pseudomonadales in AR patients’ nasal cavities and normal persons’ were big, and the differences had statistical significance. Comparison of Tag quantitative analysis showed that the amount of Gallic Streptococcus, Neisseria and halophilic Aeromonas in nasal cavities of AR patients were higher than those of normal persons(P<0.001). While the amount of Corynebacterium diphtheriae in nasal cavity of AR patients was significantly lower than that of normal persons(P<0.001). There were no significant difference in the species and quantities of bacteria of samples comparisons within each group(P>0.05).Conclusion: There is remarkable difference between the micro environment flora in the nasal cavity of AR patients and that of normal persons analyzed by 16 S rDNA sequence. The amount of Gallic Streptococcus, Neisseria and halophilic Aeromonas in nasal cavities of AR patients were significantly higher than those of normal persons(P<0.001). While the amount of Corynebacterium diphtheria was significantly lower than that of normal persons(P<0.001).The difference of microecological bacteria in the nasal cavity of AR patients and normal persons may be related to the occurrence of AR. Study Ⅲ The research to study the role and mechanism of the screened bacteria on AR by co-culturing the screened bacteria and the induced immature human peripheral blood dendritic cells.In the second study, we have screened the significantly different bacteria between AR patients and normal persons, namely C(Corynebacterium pseudodipheriticum) and S(Streptococcus gallolyticus). In the third study, we separated C and S, then co cultured with induced human peripheral blood derived iDCs to find out the influences of C and S on the differentiation and maturation of iDCs and the balance of Th1 and Th2.Methods: This study was divided into five groups, iDCs, C, CS, S and mDCs group. First of all, separate out CD14+ cells of PBMC in human peripheral blood using Human CD14 MicroBeads. Then induce the CD14+ cells to immature dendritic cells(iDCs) by application of immature dendritic cells inducing medium for 6 days. Next, iDCs group was the induced immature dendritic cells cultured using 1640 culture medium for another 48 hours. C, CS and S group were co-cultured the inactivated C, CS, and S bacterium with the induced immature dendritic cells for 48 h. mDCs group was the induced immature dendritic cells cultured using culture medium with TNF-α for 48 h to stimulate the iDCs to mDCs. We detected the amount of CD80+ and CD86+ cells in every group using flow cytometry analysis, which stand for the amount of mature dendritic cells. We measured the secretion level of IL-12 in culture medium every group by ELISA. We observed the morphology of dendritic cells in every group under field emission scanning electron microscope. Finally, cells collected from the above five groups were co cultured with CD4+T lymphocyte with the ratio of 1:20, 1:10 and 1:5 for 48 h. The secretion of IFN-γ and IL-4 in every group were evaluated by ELISA. The proliferation of CD4+T lymphocytes in every group were detected by MTT method.Results: The results of flow cytometry analysis showed that the amount of CD80+ cells and CD86+ cells in the S group was significantly higher than that in iDCs, C and CS group(P<0.05); however, no significant difference with mDCs group. ELISA testing results showed that the secretion level of IL-12 in S group compared with iDCs, C and CS groups in the culture medium was decreased, and there was statistical significance(P<0.05); no significant difference with mDCs group. Under the field emission scanning electron microscope the cells’ surface of S group and mDCs group performed a large amount of irregular dendritic protrusions, just like the surface of typical matural dendritic cells; while cells’ surface of the other groups has a relatively small amount of protrusions. After co cultured with CD4+T lymphocytes IFN-γ level was detected as i DCs group >C group >CS group >S group >mDCs group; there was statistical difference between C group and S group(P<0.05), no significant differences within group comparisons. IL-4 level was detected as iDCs group <C group <CS group <S group <mDCs group; there was there was statistical difference between C group and S group(P<0.05), no significant differences within group comparisons. Results showed that the proliferation of CD4+T lymphocytes of S group was significantly higher than that of iDCs, C and CS group; and there was an amount dependent relationship, when the ratio of cultured cells and CD4+T lymphocytes was 1:5, the proliferation of CD4+T lymphocyte was highest.Conclusion: Streptococcus gallolyticus co cultured with the induced immature human peripheral blood dendritic cells can significantly promote the transformation of immature dendritic cells into mature dendritic cells. The co-cultured cells have the ability to stimulate CD4+T lymphocytes proliferating,they also Inhibite the secretion level of IFN-γ and promote the secretion level of IL-4. Corynebacterium pseudodipheriticum co cultured with the induced immature human peripheral blood dendritic cells can significantly inhibit the transformation of immature dendritic cells into mature dendritic cells induced by Streptococcus gallolyticus. |
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