The Anti-rejection Effect Of Blocking B7/CD28 Co-stimulatory Pathway By RNAi In Mice Heart Transplantation

In this research project, RNAi technique 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. Furthermore, the anti-rejection effects were compared between sinomenine with immunosuppressive exhibition and the DC modified by RNAi in mice heartallo-transplantation.Part Ⅰ Establishment of heterotopic heart transplantation modelin mice.Objective To establish stable heterotopic heart transplantation model in mice. Methods The ascending aorta and pulmonary artery of donor heart were end-to-side anastomosed to abdominal aorta and inferior vena cava of the recipient respectively by microsurgical procedure. The Corry's pattern was improved on as follows: ligating all the vessels except for ascending aorta andpulmonary artery prior to cutting off the ascending aorta and pulmonary artery, direct irrigating the coronary system via ascending aorta of donor's heart, instead of vessel clip, ligating to stop the blood flow in recipient's abdominal aorta and vena cava inferior to broad the potential operative area. Results The surgical procedure successful rate during proficient period reaches to 85.7%. The time of graft harvest and recipient operation are respectively 5.51±1.10min, 56.91±5.23min, arterial anastomosis and venous anastomosis separately take 16.64±1.52min, 8.79±1.49 min. The ischemic time of graft is 36.63±3.25 min. Conclusion Hemorrhagic shock due to massive hemorrhage during operation and massive bleeding with arterial anastomosis apparently is the main cause of recipient death. The heterotopic heart transplantation is technically easier comparing with other solid organ transplantation in mice and seems to be skillfully performed after short time strictly training. The model is ideal and valuable for transplantation research.Part II The research on the effect of RNAi to knock-down CD80 and CD86 expressions and its mechanismObjective To investigate the influence of RNAi on DC surface antigen CD80 and CD86 expressions, explore the mechanism of DC modified by siRNA introducing T lymphocyte anergy. Methods Mice myeloid DC was cultured in selective medium containing necessary cytokines for DC growth in vitro. siRNA of which sequence specified to CD80 and CD86mRNA was synthesized in vitro respectively and transfected into DC. The expression levels of CD80 and CD86 mRNA and surface antigen CD80, CD86, MHCII, CDllc were assayed by semi-quantitative-RT-PCR and flowcytometry before and after siRNA transfection.The influence on DC stimulating the proliferation ability of T lymphocyte resulting from silencing effect mediated by siRNA was observed through MLC. IL-2, IFNy, IL-10 mRNA levels in MLC system were determined via semi-quantitative-RT-PCR. Results about 1.5-2><107 myeloid DCs may be harvested from each mouse through cell culture in vitro. After siRNA transfected into DC, the levels of CD80 and CD86 mRNA are significantly lower companying with reducing of the antigen CD80+ and CD86+ from 84%, 67% to 35% and 30% respectively, but no changing in antigen MHCII, CD lie. The result of MLC demonstrated that the index of stimulation to T lymphocyte of DC knocked down by siRNA significantly decreased accompanied by considerably lower IL-2, IFNy mRNA levels(P<0.01) and higher IL-lOmRNA in MLC system (PO.01). Conclusion Large quantity of myeloid DC with typically histological configuration and high levels of MHCII+ and CDllc+ may be obtained through vitro culture in selective medium which may activate naive T lymphocyte to generate immune response. Transfecting siRNA targeting to CD80 and CD86 mRNA with Oligofectamine into DC may efficiently and specifically inhibit CD80 and CD86 expression. The activation to allogenic T lymphocyte and IL-2 synthesis as well as secretion of DC knocked down by siRNA become weaker and lower which might result in immune deviation in Th cells and introduce T lymphocyte anergy.Part HI The anti-rejection effect due to blocking B7/CD28 co-stimulating pathway with RNAi in mice heart transplantationObjective To study the protective effect of the DC treated by siRNA and its anti-rejection mechanism. Methods The experimental animals were dividedinto 5 subgroups as the following: subgroup 1-treated by siRNA, subgroup 2-5 as controls, that are subgroup 2(allograft transplantation), subgroup 3 (treated with CsA), subgroup 4(isograft transplantation), and subgroup 5( DC non-treated with siRNA). On the 7th day pre-operatively, DCs modified by siRNA were transfused into recipients. The graft survivals were individually recorded and pathologically evaluated according to graft rejection grading on the 7 day post-operatively. IL-2, IFNy, IL-10 mRNA expression levels in grafts were determined by semi-quantitative-RT-PCR in same time. Results In siRNA modifying subgroup 1, survival of the grafts is significantly longer than subgroup 2 (26.67±4.08 vs. 11.00±2.10, PO.01), companying with significantly lower pathological grading of rejection than in subgroups 2 and 5(P<0.05), but no differences comparing with subgroups 3 and 4. Lower IL-2, IFNy mRNA levels with the elevation of IL-10 mRNA levels in grafts in subgroup 1 appear comparing with them in subgroups 2 and 5(P<0.01). Conclusion In mice heterotopic heart transplantation, donor-derived DCs in which molecules CD80 and CD86 have been knocked down through RNAi silencing could exhibit protective effect on grafts. This mechanism might be due to Th cell differentiation deviating to Th2. The cytokine IL-2 and IFNy secreted by Thl cell might be molecular markers to recognize post-transplantation rejection.Part IV The comparison of anti-rejection effect between sinomenine and DC modified by siRNA in mice hearttransplantationObjective To compare the anti-rejection effect of sinomenine with that of DC modified by siRNA and elucidate the anti-rejection mechanism of sinomenine. Methods To select subgroups 2 and 1 mentioned in Part III as controls, the...