| Objective:This study was designed to investigate the feasibility of inhibiting the CD80,CD86 expression in dendritic cells(DCs) derived from mice myeloid by shRNA plasmid vector targeting B7 transactivator,combined the CTLA4-Ig to block B7/CD28 co-stimulatory pathway to induce mouse T cell immune anergy.Methods:Mice myeloid derived DCs were cultured in vitro.The DCs were transfected with Lipofectamine2000 and plasmid of pB7shRNA which sequences were specified to B7-1 (CD80) and B7-2 (CD86) mRNA.The expression of surface antigen CD80,CD86,MHCII and CD11c was assayed by flow cytometry pre-and post-transfection. Real-time PCR (RT-PCR) was used to detect mRNA transcriptional levels of CD80 and CD86.The influence on DC stimulating allogenic T lymphocyte proliferation induced by pB7shRNA transfected DC was observed through mixed lymphocyte culture(MLC). Enzyme-linked immunosorbent assay(ELISA) was used to detect the IL-2,IL-4,IL-10,IFN-γlevels in MLC.Result:Average 1.5-2×107 DCs per mice were acquired in vitro.Flow cytometry identification showed both antigen of CD80 and CD86 were significantly inhibited by pB7shRNA post-transfection,from 90.429±1.967%,71.603±3.679%to 31.028±1.511%,39.288±1.913%(P=0.000), while there were no significant changes and differences of MHCII and CD11 c RT-PCR showed that both mRNA transcription of CD80 and CD86 were significantly inhibited by pB7shRNA post-transfection as well.MLC showed that the stimulation index of DCs transfected by pB7shRNA plasmid decreased significantly(P=0.000),ELISA showed the IL-2,IFN-y levels in MLC raised up while the IL-4,IL-10 levels decreased(P=0.000).Conclusion:In vitro culture of the mice myeloid DCs by selective medium is a reliable method to acquire sufficient DCs. It proves an effective way to transfect mice DCs with Lipofectamine2000.pB7shRNA Combined CTLA4-Ig groups show significantly inhibiting effect of B7 expression and IL-2,IFN-y of mixed lymphocyte culture system.
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