Role Of Lentivirus-Mediated RNA Interference Silencing CCR3 On The Function Of Mast Cells Of Allergic Rhinitis In Mice

Objectives:The expression of CCR3 gene on mast cells was specifically inhibited by recombinant lentivirus interference vector synthesized in vitro.The pathological changes of nasal mucosa and the number of mast cell infiltration in mice with allergic rhinitis were observed in vivo.Besides,to compare the difference of CCR3 gene expressed on mast cell and medium released from mast cell in bone marrow,peripheral blood and nasal mucosa.All these measures above were to investigate the effect of silencing CCR3 gene on mast cell function and lay a foundation for gene therapy of allergic rhinitis.Methods:Balb/c mice were randomly divided into four groups: normal control group,allergic rhinitis group,CCR3 shRNA treatment group and controlshRNA treatment group.The murine allergic rhinitis model was induced after sensitization and challenge by ovalbumin(OVA).The recombinant lentivirus vector targeting CCR3 gene synthesized in vitro was applied to allergic rhinitis model in mice by intranasal administration.Nasal symptoms of mice in each group were assessed in 30 minutes after the last nasal stimulation.Then,the bone marrow,peripheral blood and nasal mucosa from each group of mice were collected.The level of CCR3 mRNA in bone marrow,peripheral blood and nasal mucosa in each mice were measured using realtime fluorescence quantitative PCR.The level of CCR3 expression in bone marrow,peripheral blood and nasal mucosa in each mice were detected using Western Blotting.H&E staining and toluidine blue staining were used to detect the histology changes of nasal mucosa and the infiltration of mast cells in nasal mucosa,respectively.At the same time,the morphology of nasal mucociliary was observed by electron microscopy.Finally,the levels and change of histamine,tryptase and PGD2 in the bone marrow,peripheral blood and nasal mucosa were determined using commercial ELISA kits.Results:(1)After the last challenge of nasal cavity,the symptom scores of the CCR3 shRNA-treated group were lower than those of the allergic rhinitis group and the controlshRNA-treated group.(2)Real-time fluorescence quantitative PCR and western blotting indicated that CCR3 mRNA and CCR3 protein levels in bone marrow,peripheral blood and nasal mucosa were signifcantly overexpressed in allergic rhinitis group compared with normal control group;however,CCR3 shRNA treatment resulted in the reduction of CCR3 mRNA and CCR3 protein levels in CCR3 shRNA treated group compared with allergic rhinitis group or controlshRNA group,respectively.(3)H&E staining showed that the pathological changes of nasal mucosa in mice transfected with specific CCR3 shRNA lentivirus vector were improved comparing with allergic rhinitis mice.(4)Toluidine blue staining showed that mast cells numbers in the nasal mucosa of mice in the CCR3 shRNA group were less than those in the allergic rhinitis group and the controlshRNA group.(5)The nasal mucosal epithelial cells in the normal control group were arranged neatly with a large amount of cilia and uniform thickness,while in the allergic rhinitis model group and the controlshRNA model group,the nasal mucosal epithelial surface secretions increased,and the cilia were exfoliated,sparse and arranged disorderly.The cilia of the CCR3 shRNA treatment group were arranged neatly and the thickness is relatively uniform.(6)In mouse bone marrow,peripheral blood,and nasal mucosa,Elisa analysis found that the histamine and tryptase levels of the CCR3shRNA-treated group were lower than those of the allergic rhinitis group.The indexes of mice transfected with the lentivirus-mediated controlshRNA showed no significant changes compared with the allergic rhinitis group.Conclusion:By using the method of RNA interference,lentivirus vector targeting the CCR3 gene in mast cells dripped into the nasal mucosa and then could down-regulate the CCR3 expreesion in allergic rhinitis mice mucosa.Thereby effectively inhibited the migration,infiltration and degranulation of mast cells in local tissues,and alleviated the inflammation of allergic rhinitis mice.