Recombinant Adenovirus Carrying Mouse CCR7 Gene Transfect Immature Dendritic Cells Derived From Mouse Bone Marrow

Background and objectiveAllogeneic skin transplantation is the most effective method in the early coverage of extensive burn wounds.However,the host versus graft reaction(HVGR) after transplantation leads to irreversible rejection about 3 weeks later,which limits the transplantation.And there isn't any effective treatment to induce immune tolerance after transplantation.Dendritic cells(DC) are the most potent professional antigen-presenting cells(APC) in vivo, which play critical roles in immune reponse.Dendritic cells differentiate from immature dendritic cells(imDC) to mature dendritic cells(mDC).In the process, morphological characteristics, surface markers and biological functions in DC have changed accordingly.The most distinguished ability of imDC is inducing T cell anergy and promote alloantigen-specific tolerance,which has been verified in our last National Natural Science Foundation of China(NSFC) subjest(No.30271341). Based on the advantages,combined transplantation using imDC or genetically-modified imDC have seen satisfying results in kidney,liver,heart,pancreatic islet and small intestine transplantation.The phenotypical changes that take place during maturation include the blunting of the endocytic ability and the up-regulation of MHCs and costimulatory molecules that eventually transform these DC in potent APC.Maturation takes place concomitantly with the migration of the DC from their niches in the peripheral tissues to the lymph nodes where they arrive through the lymphatics.In this regard, during maturation the expression of the chemokine receptor-7(CCR7) is up-regulated.Through interaction with ligand MIP3β(CCL19)and SLC(CCL21),CCR7 guides the migratory DC to the nodes,presents antigens to naive T cells and activates immune response. Recent studies have also showed that CCR7 plays an important role in cancer metastasis.Such function of CCR7 is kind of like oriented migration in DC.If DC become more matured,they express more CCR7 ,the immune system will present stronger response.Otherwise,immune tolerance is induction.However,imDC lack the expression of CCR7 and direct less migratory DC to lymph nodes.Long time for residing in non-T cell area put imDC into high risks sensing of"danger signals"(tissue damage,inflammatory cytokines, or pathogens) ,which starts the differentiation of maturation and change the important tolerance inducing functions of imDC.Base on the the analysis above, the present study was designed to build expression of CCR7 in imDC through the construction of recombinant adenovirus carrying mouse CCR7 gene,which will afford imDC high chemotactic ability possessed by mDC, inhibit the maturation of imDC in peripheral tissues and guarantee the induction of immune tolerance of imDC in the early coverage of extensive burn woundsMethods1. The CCR7 gene was amplified by RT-PCR from thymus of mouse ,after sequencing,the fragment was cloned to T vector and subcloned to pAdTrack-CMV.The linearized shuttle plasmid was homogenously recombined with pAdeasy-1 in BJ5183.The candidate clone was further analyzed by restriction endonuclease digestion.Then the recombined plasmid was transfected into HEK293 cells for packaging,PCR and amplifying.Infection titer was monitored by green fluorescent protein (GFP) expression.2. Mouse marrow imDC isolated from BALB/c was induced, proliferated by rmGM-CSF and rmIL-4, and harvested after 6days;according to green fluorescence protein(GFP) after transfection with recombinant adenovirus we produced, the transfection efficiency was detected and the expression change of CCR7 in imDC in both mRNA and protein level was also detected with reverse transcription-polymerase chain reaction(RT-PCR) and western blot method.Results1.The recombinant adenovirus vector contaning mCCR7 was constructed successfully and the titer was about 1×109U/ml.2.The cells we cultured displayed typical morphlogical features of imDC, bigger body with dendrites were observed under microscope and GFP on the cells transfected with adenovirus were observed under fluorescent microscope.3. Recombinant adenovirus we constructed can transfected imDC with high transfection efficiency up to 70%.The expression of mouse CCR7 in imDC infected by the virus was obviously increased than the control in both mRNA and protein level.ConclusionRecombinant adenovirus carrying mouse CCR7 gene we constructed can effectively express CCR7 gene compared with the control,therefore,CCR7 has been cloned into imDC successfully. The expression of CCR7 in imDC was obviously increased in in both mRNA and protein level,which lays a solid foundation for further chemotaxis study in imDC.