Research Of Immunotolerance To Murine Cardiac Allogr-oft Induced By Combined Application Of CTLA4-Ig And RNAi To Block The B7/CD28 Costimulatory Pathway

Objective:To observe the anti-rejection effect induced by combined application of CTLA-Ig and DC treated by shRNA to block the B7/CD28 costimulatory pathway in mice heart transplantation and analysis the anti-rejection mechanism.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. And, the expression level of surface antigen CD80,CD86,CD11c,MHCⅡwere measured by flow cytometry before and after pB7shRNA transfecting DC; Established a mouse model of heterotopic heart transplantation. Six groups were assigned according to the treatment:group A(control group), group B(treated with nontransfected DC),group C(treated with pB7shRNA transfecting DC), group D(treated with CTLA4-Ig),group E(treated with nontransfected DC and CTLA4-Ig), group F(treated with pB7shRNA transfecting DC and CTLA4-Ig). Observed cardiac allograft survival time, assessed pathological grade of acute rejection on the 7 day of post-operation, at the same time the IL-2mRNA expression level in grafts were determined by real time quantitative PCR.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 posttransfection, from 93.57%,70.07% to 30.83%,40.06%, while there were no significant changes and differences of MHCII and CD11c antigen. Comparing with the group A(control group) and group B (treated with nontransfected DC), the graft survivals were significantly longer in the group F treated with pB7shRNA transfecting DC and CTLA4-Ig(MST:25 vs 8 and 8 days,P=0.001), companying with lower pathological grade of graft rejection(x2=30.602, P=0.000) and lower IL-2 mRNA expression level in grafts (P=0.000). There exists positive correlation between the IL-2 mRNA expression level and pathological grade of graft rejection (r=0.942, P=0.000)Conclusion:With the method of pB7shRNA plasmid vector transfecting DC, B7 expression will be silenced efficiently. For mice heart transplantation, transfusing donor's pB7shRNA interfered DC into recipients before operation and combining application of CTLA-Ig after operation can creat anti-rejection effect and the mechanism might be:the blockade of B7/CD28 co-stimulatory pathway by B7shRNA interfered DC and CTLA-Ig will induce the T lymphocytes of recipients anergy.