输注转染MyD88siRNA基因的供者树突状细胞延长小鼠移植心存活时间

目的 探讨输注供者来源的转染了髓样分化因子88(MyD88)siRNA基因的树突状细胞(DC)在延长同种小鼠移植心存活时间中的作用及机制.方法 以脂质体为载体,将化学合成的MyD88siRNA导入BALB/c小鼠(供者)骨髓来源的DC中,制备转染MyD88siRNA基因的DC(MyD88siRNA-DC).随机将27只C57BL/6小鼠(受者)平均分为磷酸盐缓冲液(PBS)对照组、培养8 d的DC(Day8-DC)组及MyD88siRNA-DC组,分别将PBS、Day8-DC及MyD88siRNA-DC输注至受者体内.于输注后第7、14和21天时应用免疫双荧光染色法观察供者DC在受者脾脏内的存活情况;混合淋巴细胞反应(MLR)测定受者脾脏内T淋巴细胞对供者同种抗原的反应性.另取27对供、受者(BALB/c小鼠和C57BL/6小鼠),通过袖套管技术建立颈部异位心脏移植模型,随机平均分为PBS对照移植组、Day8-DC移植组及MyD88siRNA-DC移植组,各组于移植前7 d分别经受者门静脉注射0.5 ml PBS、2.0× 106个Day8-DC及2.0× 106个MyD88siRNA-DC.于输注后第7天,观察各组移植心的存活时间;病理检查观察排斥反应程度;酶联免疫吸附试验测定受者血清巾Th1及Th2型细胞因子[γ干扰素(INF-γ)、白细胞介素(IL)-12、IL-4和IL-10]水平的变化.结果 MyD88siRNA-DC在受者脾脏内的存活时间明显延长,MyD88siRNA-DC组受者脾脏内T淋巴细胞对供者抗原的反应性最低(P＜0.01).PBS对照移植组、Day8-DC移植组及MyD88siRNA-DC移植组移植心的存活时间分别为:(6.67±1.37)d、(13.67±2.25)d和(24.50±4.42)d,与PBS对照移植组相比,Day8-DC移植组移植心存活时间延长(P＜0.01),而MyD88siRNA-DC移植组移植心存活时间较Dby8-DC移植组进一步延长(P＜0.01);MyD88siRNA-DC移植组移植心排斥反应病理分级最低,受者血清中INF-γ和IL-12水平显著降低(P＜0.01),而IL-4和IL-10水平明显升高(P＜0.01).结论 输注转染MyD88siRNA基因的供者DC能够延长同种小鼠移植心的存活时间;其机制可能与诱导受者Th1/Th2免疫偏移及形成供、受者微嵌合状态有关.

Pretreatment of donor dendritic cells with MyD88siRNA to induce tolerance in mouse allograft recipients

Objective To explore the effects of donor dendritic cells (DC) treated with MyD88siRNA in tolerance induction in mouse allograft recipients.Methods MyD88siRNA was synthesized chemically and transfected into DC derived from BALB/c bone marrow by RNAi-mate.The DC modified with MyD88siRNA,named MyD88siRNA-DC,were injected into the recipient C57BL/6 mice.The existence of the donor MyD88siRNA-DC in the recipient animal spleens was studied by double-immunofluorescence staining.The responsiveness of the recipient spleen T cells to the donor alloantigen was determined by mixed lymphoeyte reaction(MLR).The cervieal heterotopic heart transplantation model was established with "cuff" technique 7 days after iniection and the cardiac allograft survival time was observed.Pathological analysis was performed and the levels of cytokines in the serum were determined using ELISA. Results The survival rate of the donor-derived MyDB8siRNA-DC in the recipient spleens was higher than that of the donor derived Day8-DC.The donor-derived MyD88siRNA-DC induced alloantigen-specific T-cell hypo-responsiveness.The cardiac allograft survival time in the MyD88siRNA-DC treated group was longer than that in the Day8-DC group and PBS treated group [(24.50±4.42) days vs (13.67±2.25) days and (6.67±1.3) days,(P＜0.01)].Pathological grade of rejection was significantly lower (P＜0.01).In the MyD88siRNA-DC treated group,the levels of IL-12 and IFN-γ in the serum were decreased significantly (P＜0.01),but the levels of IL-4 and IL-10 in the serum increased significantly (P＜0.01).Conclusions The injection of the donor-derived MyD88 siRNA-DC can lcad to donor-specific tolerance in transplant recipients.The polarization of Th2 response and chimerism of recipient may play important roles on immune tolerance to cardiac allografts.