The Research On The Construction Of B7shRNA Plasmid Vector And Its Anti-rejection Effect In Mice Heart Transplantation

In this research project, B7 shRNA plasmid vector was constructed and was used to modify myeloid DC in that way which the CD80 and CD86 expressions on it were inhibited to block B7/CD28 co-stimulatory pathway in order to explore the mechanism of T lymphocyte anergy. The protective effect to allograft and anti-rejection mechanism in heart allo-transplantation due to donor derived DCs underwent CD80 and CD86 knock down by RNAi were investigated.Part I Construction of B7shRNA Plasmid Vector and InvitroResearch on its Effect of Blocking B7/CD28 Co-stimulatory PathwayObjective: To construct small hairpin RNA(shRNA) plasmid vector targeting B7 molecular of mice dendritic cells(DC) and analysis its effect on blocking B7/CD28 co-stimulatory pathway and inducing T lymphocytes anergy .Methods: Mice myeloid DC were cultured invitro. shRNA plasmid vector pCD80shRNA, pCD861shRNA , pCD862shRNA were constructed and then transfected DC respectively, correspondly, The change of surface antigen CD80, CD86 expression levels were measured by flowcytometry. pB7shRNA plasmid vector targeting both CD80 and CD86 was constructed using pCD80shRNA and pCD862shRNA. The mRNA expression level of CD80, CD86 and the expression level of surface antigen CD80, CD86, CD11c, MHCⅡ were measured by real time quantitative PCR and flowcytometry before and after pB7shRNA transfecting DC. The influence on DC stimulating allogenic T cell proliferation induced by pB7shRNA transfected DC was observed through MLC. IL-2 mRNA levels in MLC were determined by real timequantitative PCR. Results: The CD80 antigen expressing level declined from 93.42% to 31.05% after the pCD80shRNA transfecting DC, while CD86 declined from 78.70% to 44.26% and 37.90% after the pCD861shRNA and pCD862shRNA transfecting DC respectively. The pB7shRNA plasmid vector targeting both CD80 and CD86 was constructed successfully using pCD80shRNA and pCD862shRNA. After the pB7shRNA transfecting DC, the CD80> CD86 mRNA expression were inhibited at the rate of 77.5% and 61.5% respectively, companying with reducing of the surface antigen CD80+ and CD86+ from 93.42%, 78.70% to 30.05% and 34.76% respectively, but no changing in surface antigen of MHCII, CD lie. The MLC result showed that the index of stimulation to allogenic T cell was declined significantly after the pB7shRNA transfecting DC (P<0.0\), and the IL-2 mRNA levels in MLC was inhibited at the rate of 80.48%. Conclusion: The construction of pB7shRNA plasmid vector applied a stable vector for long-time RNAi research on B7 molecules of mice DC. With the method of pB7shRNA plasmid vector transfecting DC, B7 expression will be silenced efficiently and the B7/CD28 co-stimulatory pathway will be blocked , the ability of DC that stimulate allogenic T lymphocytes proliferation and IL-2 secreation will be knocked down ,thus the T lymphocytes will be induced anergy.Part II The Research on the Anti-rejection Effect Induced byB7shRNA Interfered DC in Mice Heart TVansplantationObjective: To observe the anti-rejection effect of B7shRNA interfered DC in mice heart transplantation and analysis the anti-rejection mechanism. Methods: At first, the heterotopic heart transplantation model in mice was established, then the B7shRNA interfered DC of donors were were transfused into recipients 7 days before operation(group 1: DC treated by shRNA), and the four control groups were:group2: isograft transplantation, group3: allograft transplantation, group4: treated with CsA, group5: DC none treated with shRNA. The graft survivals were individually recorded and pathological grading of graft rejection were evalued 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. Results: Stable heterotopic heart transplantation model in mice was established successfully. Comparing with the group3 and group5, the graft survivals were significantly longer in the group of DC treated by shRNA (group 1),(MST: 22 vs 9 and 8 days, P<0.0\ ) companying with lower pathological grade of graft rejection( x2=36.566, PO.01 ) and lower IL-2 mRNA expression level in grafts ( 0.117 + 0. 039 vs 0.376 ±0. 068 and 0.436 ± 0. 055, P<0.0\ ) . There exists positive correlation between the IL-2 mRNA expression level and pathological grade of graft rejection (r=0.931, P <0.01) . Conclusions: Mouse heterotopic heart transplantation model is ideal and valuable for transplantation rejection research. For mice heart transplantation, transfusing donor's B7shRNA interfered DC into recipients before operation can creat anti-rejection effect and the mechanism might be: the blockade of B7/CD28 co-stimulatory pathway by B7shRNA interfered DC will induce the T lymphocytes of recipients anergy. IL-2 might be molecular makers to recognize post-transplantation reaction.