| In this study, mouse bone marrow-derived dendritic cells (DCs) were infected by CD40-RNA interfering (RNAi) lentiviral vector to inhibit the expression of CD40. Then we explored the mechanism of T lymphocyte incompetence induced by the DCs through blocking CD40/CD40L costimulatory pathway and investigated the anti-rejection mechanism of tolerogenic DCs with low expression of CD40 and FTY720 in allogeneic heart transplantation.Part 1 The research on the lentivirus mediated RNAi to knockout CD40 expression of dendritic cells and the mechanism of T lymphocyte incompetence induced by the DCsObjective:To construct the mouse CD40-RNAi lentiviral vector and prepare tolerogenic DCs (Tol-DCs) with low expression of CD40. To explore the mechanism of inducing T lymphocyte incompetence by blocking CD40/CD40L costimulatory pathway. Methods:We cultured mouse bone marrow-derived DCs in vitro and constructed the mouse CD40-RNAi lentiviral vector. Five groups were assigned according to the treatment:nontreated group, NC-GFP-lentivirus (LV) infected DC group and three CD40-RNAi-LV infected DC groups (24h,48h,72h after infection). Fluorescence real-time quantitative PCR and flow cytometry were used to analyze the expression of CD40mRNA and DC surface antigen CD40, CD11c, MHCII before and after infection. Mixed lymphocyte reaction was performed to study the impact on the capacity of allogeneic T lymphocyte proliferation stimulated by CD40-RNAi lentiviral infected DCs. Results:A mouse CD40-RNAi lentiviral vector was builded successfully. The lentiviral titer was 8E+9 TU/Ml. Compared to noninfected group, CD40mRNA inhibition rates of three CD40-RNAi-LV infected DC (24h,48h,72h after infection) groups (41.8%,80.9%,83.0%) were higher respectively (PO.05). The CD40mRNA inhibition rate of 48h. after infection was significantly higher than that of 24h after infection. There was no difference of CD40mRNA inhibition rate between 48h and 72h after infection (P>0.05). Compared to noninfected group(74.37%±4.08%), CD40 protein expression of 48h after infection (40.07%±4.03%) and 72h after infection (26.32%±3.60%) were both lower (P＜0.05). CD40 protein expression of 72h after infection was significantly lower than that of 48h after infection (P<0.05). There was no difference of CD40 protein expression among noninfected, NC-GFP-LV infected (72.45%±2.65%) and CD40-RNAi lentivirus infected DCs 24h after infection (70.83%±4.87%)(P>0.05). Both CDllc and MHC II protein expression on DCs among the five groups had no significant change (P>0.05). Mixed lymphocyte reaction show that, lymphocyte stimulation index (SI) of CD40-RNAi-LV infected DC group (1.381±0.442) was lower than that of noninfected group (2.444±0.485) (P<0.01). There was no significant difference of SI between noninfected group and NC-GFP-LV infected DC group (2.294±0.367) (P>0.05). Conclusion:A stable and reliable vector, the mouse CD40 RNAi lentiviral vector, was established successfully for the future studies in vivo. While DCs were infected by CD40-RNAi lentiviral vector, CD40 protein expression was inhibited significantly. The DCs could hamper the activation of allogeneic T lymphocyte by blocking CD40/CD40L costimulatory pathway and induce T cell anergy.Part 2 The research on the anti-rejection effect of DCs infected by CD40-RNAi lentiviral vector combined with FTY720 in mouse heart transplantation Objective:To observe the different anti-rejection mechanism of administering donor DCs infected by CD40-RNAi lentiviral vector and FTY720 in mouse allogeneic abdominal heart transplantation. Methods:We established a mouse model of heterotopic abdominal heart transplantation. Five groups were assigned according to the treatment:control group, noninfected DC group, FTY720 administered group, lentivirus infected DC group and lentivirus infected DC with FTY720 administered group. We observed cardiac allograft survival time, assessed pathological grade of acute rejection 7d after heterotopic abdominal heart transplantation and analyzed CD4+CD25+regulatory T cells (Tregs) in peripheral blood before and after transplantation by flow cytometry. Results:A mouse model of heterotopic abdominal heart transplantation was builded successfully. Compared to control group(8d) and noninfected DC group (9d), cardiac allograft median survival time of CD40-RNAi lentivirus infected DC group (18d) was significantly longer (P<0.01). The pathological grade of acute rejection (7d after transplantation) in CD40-RNAi lentivirus infected DC group was lower than that of control group significantly (P＜0.01). Compared to control group((2.16%±1.00%) and noninfected DC group(3.49%±1.49%), CD4+CD25+Tregs in peripheral blood 3 days after transplantation(24.2%±6.06%) and 7 days after transplantation(22.8%±3.34%) in CD40-RNAi lentivirus infected DC group were both significantly increased (P<0.01). Compared to lentivirus infected DC group, cardiac allograft median survival time of lentivirus infected DC with FTY720 administered group (26d) was significantly longer (P<0.01). However, there was no significant difference of pathological grade of acute rejection and the level of Tregs in peripheral blood before and after tansplantation between the two groups (P>0.05). Conclusion:It was a feasible model of heterotopic abdominal heart transplantation in mice to study the rejection after tansplantation. The mechanism of the anti-rejection in mouse allogeneic heart transplantation may due to Tol-DCs with low expression of CD40 from the donor blocked CD40/CD40L costimulatory pathway and stimulated the generation of Tregs, inducing T lymphocyte impotence. Blocking CD40/CD40L costimulatory pathway combined with FTY720 was a more effective approach to induce immune tolerance, compared to blocking CD40/CD40L costimulatory pathway only.
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