Effection Of Dendritic Cells Modified By The Recombinant Adenovirus Of CTLA4Ig And CCR7 In Treating Asthma

PARTâ… THE CLONE OF CCR7 GENE OF MOUSEObjective:To clone the CCR7 gene from the lung of mouse. Methods:Total RNAs were isolated from the lung of mouse.The CCR7 gene was amplified by RT-PCR using the primers based on the published sequence of CCR7.The PCR products were purified by gel extraction and cloned into pMD-19.Analyzed the recombinant plasmids pMD-19-CCR7 through sequencing and sequence comparison with CCR7 gene in GeneBank.If found mutation basic groups which would develop site-specific mutagenesis,it was necessary to repair them.Result:The CCR7 gene was cloned.2 basic groups in CCR7 gene order by the sequence comparison were revealed,while that did not develop site-specific mutagenesis and resulted in encoding alteration,so it was not necessary to repair.Conclusion:The CCR7 gene was cloned successfully in mouse which did not develop site-specific mutagenesis.PARTâ…¡CONSTRUCTION OF THE RECOMBINANT ADENOVIRUS: ADCTLA4IG,ADCCR7Objective:To construct the recombinant adenovirus: AdCTLA4Ig,AdCCR7,including CTLA4Ig,CCR7 genes with the newest AdEasy system and control AdGFP.Methods:1.The CTLA4Ig,CCR7 genes were coloned to the shuttle plasmids pAdTrack-CMV.The linearized shuttle plasmids were co-transformed with adenovirus backbone vector to E.coli BJ5183 .Before transfection the recombinant adenoviral plasmids were digested with Pacâ… .Then the digested recombinant adenoviral plasmids were transfected to 293 cells with Lipofectamine 2000.The generations of the recombinant adenovirus were monitored by GFP expression.The presences of recombinant adenoviruses were further confirmed by RT-PCR.Result:The straps of 4.5Kb or 3Kb and near 30Kb appeared after digested with Pacâ… .The result showed that the correct recombinant adenoviral plasmids were constructed.After transfected into 293 cells,the green fluorescence was observed.Conclusion:The recombinant adenovirus of AdCTLA4Ig,AdCCR7 and controls AdGFP were constructed.PARTâ…¢DENDRITIC INFECTED WITH ADCTLA4IG AND ADCCR7Objective:The dendritic cells were isolated from the bone marrow of the mice to set up the primary culture method in vitro.To identify the factors that influenced the mature of the dendritic cells and the expression efficiency of CTLA4Ig and CCR7.To construct the dendritic cell vaccines infected by AdCTLA4Ig and AdCCR7.Methods: The bone marrow cells were collected from the tibial and femurs of the mice,and cultured with GM-CSF and IL-4 in vitro for 7 days.The growth and morphology of the cells were observed by light microscope.The dendritic cells were identified by FACS and infected with the recombinant adenivirus,and then cocultured with OVA.The maturation of the dendritic cells was identified by FACS.The expression of intracellular report gene and the growth of the dendritic cells were detected by fluorescence microscope.The expressions of purpose genes were detected by FACS and laser scanning confocal microscope .Results: 50% of the cells were showed the phenotype of CD11c and were definited as the dendritic cells.About 75% of the cells coexpressed CTLA4Ig and CCR7 protein by laser scanning confocal microscope . The expression of costimulatory molecules CD40/CD80/CD86 on the dendritic cells were decreased compared to matured dendritic cells.Conclusion: Dendritic cells infected with AdCTLA4Ig and AdCCR7 could express CTLA4Ig and CCR7 proteins.The expression of costimulatory molecules CD40/CD80/CD86 on the dendritic cells were decreased.PARTâ…£THE STUDY ON THE TREATMENT OF ASTHMA BY DCS MODIFIED WITH ADCTLA4IG AND CCR7Objective: To investigate the therapeutic effection of AdCTLA4Ig/AdCCR7-DCs on asthmatic airway inflammation and the cytokine secreted by T lymphocytes. Methods: SPF level BALB/c mice were randomly divided into 4 groups: healthy na?ve mice sensitized/challenged with normal saline(NS),OVA sensitized/challenged mice, OVA sensitized/challenged mice intravenous injected with AdCTLA4Ig/AdCCR7-DCs or AdGFP-DCs two hours before challenging.The airway inflammation was determined by histopathological examination.Cytokines were measured by ELISA.The levels of CCR7 were analyzed by immunofluorescent.Results: Our data showed that compared to the mice intravenous injected with AdGFP-DCs(control), intravenous injecting with AdCTLA4Ig/AdCCR7-DCs could protected the mice from the subsequent induction of asthmatic airway inflammation.The eosinophil infiltration in BALF was significantly lower than the control.Compared to the AdGFP-DCs treated mice,the levels of cytokine were significantly decreased in BALF,but not in serum.Our data also suggested that intravenous injected with AdCTLA4Ig/AdCCR7-DCs,the levers of CCR7 in lung were significantly increased compared to the mice of other groups,and also kidney/intestine of the same group.Conclusions: This study demonstrated that intravenous injecting with AdCTLA4Ig/AdCCR7-DCs was able to attenuate the asthmatic airway inflammation and eosinophilia,regurate the cytokines secreting in local airway with minor systemic effects,avoiding sys tolerance.