| Background:Allergic rhinitis?AR?is a chronic respiratory inflammatory disease in the upper respiratory tract,characterized by paroxysmal repetitive sneezing,watery rhinorrhea,and nasal itching.It is caused by IgE-mediated reactions and involving a variety of immune cells and cytokines driven by type 2 helper T cells.Eosinophil?EOS?is the focus and hotspot in the study of the pathogenesis of allergic rhinitis.It is a kind of granulocyte leukocytes derived from bone marrow CD34+progenitor cells.EOS of the inflammatory sites is a sign of severity of inflammation or allergic reactions,and many studies have shown that reducing eosinophilic inflammatory response is one of the key aspects of AR treatment.The process of EOS chemotaxis,recruitment and infiltration depends on eosinophil-specific cytokines and chemokines.Eotaxin is considered to be an important immunomodulation of EOS migration to inflammation sites.A large number of studies have shown that Eotaxin is through the combination with the CCR3 receptor to recruit EOS to the inflammatory tissue and activation of them.CCR3 is a seven-fold transmembrane conjugated protein receptor,which was first found expressing on the surface of EOS cells,which belongs to the CC class of chemokine receptors.CCR3 is the sole receptor for Eotaxin..Eotaxin belongs to the only immune chemokine that recognizes the signal through CCR3.The CCR3/Eotaxin pathway participates in the process of eosinophils recruiting into the lungs in the mouse model of the asthma model,which is also involved in allergic dermatitis.So that blocking CCR3/Eotaxin pathway can provide a new method for the treatment of allergic diseases.In this study,we constructed CCR3 gene knockout mice model by appling gene knockout technology and carried out in vivo and in vitro experiments.In vitro experiments,culture eosinophils from bone medulla of CCR3gene knockout mice.The proliferation,maturation and apoptosis of EOS were observed.In vivo experiments,we successfully constructed the allergic rhinitis model induced by OVA sensitization,and observed the clinical manifestations of sneezing and watery rhinorrhea in CCR3+/+and CCR3-/-mice,observed the growth and development of mice.The changes of CD34+eosinophilic progenitor cells and the changes of mature EOS in the bone marrow,peripheral blood and nasal mucosa of mice were detected.We also examined the changes of EOS degeneration and the effects of knockout CCR3 gene on Th1/Th2 Immune balance.Through the above study,we studied the effect of CCR3 on eosinophils in allergic rhinitis mice induced by OVA sensitization,so as to explore a new method for the prevention and treatment of allergic rhinitis.Part? Changes in eosinophils in mice with CCR3 knockout miceObjective:The mouse model of CCR3 knockout was constructed by gene knockout.culture.The eosinophils from bone medulla were cultured in vitro and detected changes characteristics of eosinophils between CCR3-/-mice and Balb/c wild type mice Of the changing.Methods:The mouse CCR3 gene was knocked out by homologous recombination technique.The bone marrow primitive cells from CCR3-/-mice were cultured in vitro to induce the differentiation of bone marrow cells into eosinophils.The eosinophils were observed under inverted microscope Granulocyte proliferation,qRT-PCR detection of eosinophil degranulation protein m RNA expression,Annexin V-FITC/PI double staining method to detect EOS cell apoptosis.Results:1.Between days 10 and 14,the proliferation of EOS in CCR3 knockout group was significantly lower than that in normal Balb/c wild type control group?P<0.01?.2.The results of EOS cell apoptosis showed that the cell culture medium with or without IL-5,EOS cell apoptosis rate in CCR3 gene knockout group was higher than the normal Balb/c wild type control group,The difference was statistically significant?P<0.0001?.3.The expression of ECP,EPO and MBP mRNA was detected by qRT-PCR.The results showed that the expression of ECP,EPO and MBP mRNA in primary culture of CCR3 knockout group was significantly lower than that of normal Balb/c Wild type control group,the difference was statistically significant?P<0.05?.Conclusions:CCR3 gene can regulate the growth and development of eosinophils,knockout CCR3 gene can inhibit the proliferation and maturation of EOS,and promote its apoptosis.Part II:Effect of CCR3 gene knockout on eosinophils in allergic rhinitisinduced by OVAObjective:To construct a CCR3 knockout mouse model to study the effect of CCR3knockout on eosinophils in mice with allergic rhinitis induced by OVA.Methods:The mutant CCR3 gene was knocked out by gene knockout,and the allergic rhinitis mice were induced by different doses of ovalbumin?OVA?.The clinical manifestations of mice were observed.The infiltration of EOS in nasal mucosa of mice and the pathological changes of important tissues and organs of liver and kidney were detected by HE staining.The number of EOS and CD34+progenitor cells in bone marrow,peripheral blood and nasal lavage fluid were measured by Wright's staining.The expression of CCR3 and EOS granule mRNA in bone marrow,peripheral blood and nasal lavage fluid were detected by real-time fluorescence quantitative polymerase chain reaction?PCR?.The level of EOS granule protein in bone marrow,peripheral blood and nasal mucosa was detected by IHC method.The changes of IFN-?,IL-4 and IgE in peripheral blood and nasal cavity of mice were detected by flow cytometry?FCM?.The changes of eosinophils and the apoptosis of mice were detected by flow cytometry.Results:1?CCR3-/-mice had no significant nasal symptoms compared with Balb/c wild type mice under the stimulation of allergen.Organ pathological structure did not change,nasal mucosal pathological changes were significantly reduced.2?Wright's staining test showed that the proportion of eosinophils in bone marrow,peripheral blood and nasal lavage fluid of CCR3-/-allergic rhinitis mice was significantly lower than that of CCR3+/+allergic rhinitis mice?P<0.05?.?3?Comparison of CCR3-/-allergic rhinitis mice with CCR3+/+allergic rhinitis mice,eosinophil degranulation protein in bone marrow,peripheral blood and nasal lavage fluid:cationic protein?ECP??MBP?and peroxidase?EPO?were significantly decreased?P<0.05?.?4?After stimulating by OVA,the bone marrow-derived cells were cultured to detect the number and apoptosis of eosinophils in vitro,CCR3-/-mice had lower eosinophil numbers than CCR3+/+mice,with a higher apoptotic rate than CCR3+/+mice;The number of CD34+eosinophilic progenitor cells in bone marrow,peripheral blood and nasal lavage fluid was lower than that of CCR3+/+mice.?5?Compared with the control group,the level of IFN-?in serum of OVA-sensitized allergic rhinitis mice was significantly decreased,and the levels of IgE and IL-4 were significantly increased.Compared with CCR3+/+allergic rhinitis mice,the levels of IFN-?,IgE and IL-4 in CCR3-/-mice with allergic rhinitis increased,suggesting that CCR3 gene was associated with the development of allergic rhinitis,in the process of Th1 cytokines and Th2 cytokine imbalance has a very important role.Conclusions:CCR3 gene knockout,does not affect the growth and development of mice,can inhibit the differentiation of eosinophils development,migration and tissue infiltration.At the same time,CCR3 gene may be one of the reasons of imbalance between Th1cytokines and Th2 cytokines in allergic rhinitis... |
PDF Full Text Request