The Research On RNAi Inhibiting Effect To Co-stimulatory Pathway In Mouse Myeloid Dendritic Cell

Objective:This study was designed to investigate the feasibility of inhibiting the CD80,CD86 expression in dendritic cells(DCs) derivied from mice myeloid by shRNA plasmid vector targeting B7 transactivator to regulate B7 expression for suppressing immune rejection.Methods:In vitro culture of the mice myeloid DCs was performed by selective medium containing essential cytokines for its growth.The DCs were transfected with Lipofectamine2000 and plasmid of pB7shRNA which sequences were specified to B7-1(CD80) and B7-2(CD86) mRNA.Also,the expression of CD80,CD86,MHCⅡand CD11c antigen 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,and flow cytometry to detect CD80 and CD86 antigen level after tranfection.Result:Average1.5-2×10~7 DCs per mice were acquired in vitro.Flow cytometry identification showed both antigen of CD80 and CD86 were significantly inhibited by pB7shRNA posttransfection,from 92.812±1.377%,70.510±1.947%to 31.137±1.702%,40.51±1.55%(P=0.000),while there were no significant changes and differences of MHCⅡand CD11c antigen.RT-PCR showed that both mRNA transcription of CD80 and CD86 were significantly inhibited by pB7shRNA posttransfection as well(P=0.000).Mixed lymphocyte culture showed that the stimulation index of DCs transfected by pB7shRNA plasmid decreased significantly (P=0.000).The mRNA transcription of IL-2 of mixed lymphocyte culture system were significantly inhibited to 25.675±1.521%(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 groups show significantly inhibiting effect of B7 expression and IL-2 of mixed lymphocyte culture system.