Effect Of Rnai Silencing CCR3 Gene On Mouse Mast Cells In Vitro

Objectives:To synthetize and appraise lentiviral shRNA vector carrying mice CCR3 gene,mice bone marrow-derived mast cell CCR3 gene was silenced by interference vector,and the effect of CCR3 gene on the proliferation,apoptosis and degranulation of mice mast cell was further studied.To provide a theoretical basis for gene targeted treatment of allergic rhinitis.Methods:1.An ex vivo culture system was adopted to purified mast cells from mice bone marrow progenitors,cell identification was carried out by toluidine blue staining and flow cytometry.2.Synthetized three lentiviral shRNA vectors carrying mice CCR3gene: firstly,interference sequence of CCR3 was connected to PDSO19-PL RNAi lentiviral vector;Next,connection product was switched to competent cells in Bacteria;lastly,the growing monoclonal bacterial colony was identified by DNA sequencing.The correct clon by DNA sequencing was the lentiviral RNAi vector carrying target gene CCR3.3.Packaged lentivirus and measured titer: highly purified and non-endotoxic lentiviral shRNA vector and auxiliary vector were extracted,and then transfected to 293 T cells through Lipofectamine 2000,the lentiviral-rich supernatants was collected after promoting transfection,changing the solution and breeding.High titer of lentiviral was obtained and measured by condensing the supernatants.4.Testted effect of lentiviral acting on CCR3: made lentiviral and NC-lentiviral infect mast cells,and evaluated jamming effect of lentiviral vector acting on target gene by Western blot and RT-PCR.5.The CCR3 mRNA and protein expression level of mast cell were detected by RT-PCR and Western Bolt.6.To detect the influence of CCR3 on the proliferation and apoptosis of mast cells through CCK-8 and flow cytometric.7.To detect the influence of CCR3 on the chemotaxis of mast cells through Transwell.8.To detect the influence of CCR3 on the degranulation of mast cells through elisa.Results:(1)Three lentiviral vectors were identified by DNA sequencing that all DNA sequences did not contain mutation site.It proved that construction of plasmid was successful.(2)After transfecting lentiviral vector plasmid to 293 T cells,green fluorescence was obviously observed.To Package lentivirus particles in 293 T cells,after purifying,high titer of PDSO19-PL-CCR3 plasmid was received eventually.(3)Target cells were tested by western blot and RT-PCR that target gene CCR3 in three lentiviral vector were certainly down-regulated and the CCR3-shRNA2 was the most obvious.(4)The RT-PCR and Western Blot results indicate that lentiviral vector of CCR3 gene has interference effect on the level of mRNA and protein inhibition(P<0.05).(5)CCK8 and flow cytometric results indicate that silencing CCR3 inhibited the proliferation of mast cells(P<0.05),but increased the apoptosis ratio conversely(P<0.05).(6)Transwell results indicate that silencing CCR3 inhibited the chemotaxis of mast cells(P<0.05).(7)Elisa results indicate that silencing CCR3 inhibited the degranulation of mast cells(P<0.05).Conclusion:The mice CCR3 gene lentivirus shRNA vector plasmid was successfully constructed by RNAi technique,in which CCR3-shRNA2 silencing was the most efficient.CCR3-shRNA2 can effectively down-regulate the expression of CCR3 gene mRNA and protein in murine mast cells.And,it can promote the apoptosis of mouse mast cells and inhibit the proliferation,chemotaxis and degranulation of mouse mast cells.It can be considered as a target for the treatment of allergic rhinitis.