The Effect Of Dendritic Cell In Acute Rejection On Heteropic Heart Transplantation In Mice

Organ transplantation has become the best choice of end-stage organ failure, but it is far from perfect. Acute rejection (AR) is still the biggest obstacle affecting transplant success, but its mechanism is not yet fully clear, its mainly mediated by the T cell immune response. It has been confirmed that the antigen-presenting cells(APC) can induced the activation and proliferation of T lymphocytes by direct and indirect recognition,which mediate the occurrence of acute rejection. dendritic cells(DC) is the most dedicated APC in bodys, it start immune response while through the process of maturation, immature dendritic cells (iDC) low expression of MHCII molecules, with only the antigen phagocytic function, when it stimulation by danger signals,it becam mature and migrate to secondary lymphoid organs, mature dendritic cells (mDC) high expression of MHCII and costimulatory molecules CD80、CD86, co-mediated rejection. All the obove is the acute rejection mechanism theory, still need to be further confirmed by in vivo experiments. First, this project successfully established murine model of acute rejection after heart transplantation, followed detect the change of dendritic cell maturation.Model:the BALB/c and C57BL/6mice were randomly divided into three groups:①control group (BL/6→BL/6n=5);②transplantation group A (Balb/c→BL/6n=6);③transplantation group B(BL/6→Balb/c n=6).The donor heart transplant in to receptors abdomen,heart donor ascending aorta and pulmonary artery respectively Suture with the receptor abdominal aorta and inferior vena cava.Make hematoxylin-eosin staining when the graft stop working,observed the change of graft lymphocyte infiltration. Altogether heart transplantation were20cases,17cases were successfully, the success rate was85%.In Control group,there is only a small amount of graft lymphocyte infiltration,no rejection,and long-term graft survival; there are Obviously lymphocyte infiltration in transplantation group A and group B,rejection occurred in both groups,but there was no significant difference in the score and transplant rejection Graft Survival time (P <0.05).Mouse heart transplantation model technology is mature,exclusion is relatively easy to determine, is the more reliable models of acute rejection.As the C57BL/6mice has more transgenic lines,it more suitable as a receptor.Dendritic cells and maturity testimake the Paraffin sections of mouse heart first,then detected by IHC.The results showed that:there was no Obvious expression of CD11c in the control group,only see little yellow granules,while showed positive expression in the transplant grafts of both group A and B,significant higher than the control group (P<0.05),but no significant difference between the two groups (P>0.05).Prepared single cell suspensions when the graft stoped working,and then detected the expression of MHCⅡ、CD45、CD11c and CD86by FCM.The results showed that,compared with the control group and blank group,CD45was significantly increased in both group A and group B(P<0.05),while the expression of dendritic cell surface markers CD11c was significantly increased,but no significant difference (P>0.05),we also found its mature markers CD86+MHCⅡ+was significantly increased(P<0.05).qRT-PCR detection:Total RNA was extracted from cardiac graft using TRIzol according to the manufacturers’instruments, RNA was reversed transcription to cDNA by M-MLV reverse transcriptase and then amplifying IL-12, IFN-y, TNF-α and CD11c, CD80, CD86, MHCⅡ by real-time quantitative PCR using the specific primers. mRNA expression ratio of all the factors between intervention group and the non-intervention group was calculated according to the relative quantitative formula2-ΔΔCT.The results showed that:compared with the control group,the expression of IL-12, IFN-γ, TNF-α and CD11c, CD80, CD86, MHCⅡ was significantly increased in both group A and group B(P<0.05),but there was no significant difference between the two groups(P>0.05).