负向免疫调节树突细胞对心脏移植大鼠Th1/Th2类细胞因子的影响

Effect of a novel negative immunoregulatory dendritic cell on Th1/Th2 balance of cardiac allograft recipient rats

目的探讨受体树突细胞(DC)与体外光化学法(PUVA)处理的供体脾淋巴细胞共培养后,对心脏移植受体Th1/Th2类细胞因子及移植物存活的影响。方法以DA大鼠为供体,LEW大鼠为受体,建立大鼠腹部异位心脏移植模型。分离正常的供体脾淋巴细胞(SP),制备经PUVA处理的供体脾淋巴细胞(PUVA-SP)。在体外将PUVA-SP或SP与受体骨髓来源的未成熟DC共同培养,检测上述处理对受体DC分泌白介素-10(IL-10)及干扰素-γ(IFN-γ)的影响。按受体术前1周静脉输注的细胞成分将受体大鼠随机分为3组:对照组(n=7),单纯输注PBS;SPDC组(n=8),输注负载供体SP的受体大鼠DC;PUVA-SPDC组(n=8),输注负载PUVA处理的供体SP的受体大鼠DC。观察移植物的存活时间,移植术后6d检测受体血清中Th1细胞因子(IL-2、IFN-γ)及Th2细胞因子(IL-10),并取移植心脏标本作切片检查,确定排斥反应的病理分级。结果受体DC与供体PUVA-SP共培养后,其分泌的细胞因子IL-10、IFN-γ的水平分别为75.3±3.7、24.3±1.2ng/ml,明显高于同供体SP共培养后的受体DC(分别为39.0±3.1ng/ml、12.1±0.5ng/ml,P<0.01)。心脏移植术后,PUVA-SPDC组受体大鼠血清中Th1细胞因子IL-2和INF-γ的浓度分别为199.3±8.4、9.0±1.2ng/ml,明显低于对照组(分别为295.0±3.2、61.0±3.2ng/ml,P<0.01)及SPDC组(分别为559.3±35.3、69.0±2.3ng/ml,P<0.01),而其Th2细胞因子IL-10水平(58.2±0.9ng/ml)明显高于对照组(19.0±0.6ng/ml,P<0.01)及SPDC组(20.1±1.6ng/ml,P<0.01)。PUVA-SPDC组移植物排斥反应病理分级明显低于对照组,而对照组移植物排斥反应病理分级则低于SPDC组。与对照组(6.7±0.3d)比较,PUVA-SPDC组移植物存活时间(27.3±1.3d)显著延长(P<0.01),而SPDC组移植物存活时间短于对照组(5.5±0.3d,P<0.05)。结论PUVA-SPDC具有负向免疫调节特性,能够在移植物受体体内诱导Th2免疫偏移,下调移植物排斥反应。

Objective To investigate the effect of recipient derived dendritic cells （DC） loaded with 8-methoxypsoralen plus UVA irradiation （PUVA）-treated donor splenic lymphocytes （PUVA-SP） on Th1/Th2 balance and cardiac allograft rejection in recipient rats. Methods Heterotopic vascularized heart transplantation model was established in inbred DA and LEW rats. Spleen lymphocytes of DA rat were treated with PUVA. Bone marrow-derived DCs of LEW rat were co-cultured with PUVA-treated or untreated DA spleen lymphocytes （PUVA-SP or SP）, and the productions of IFN-γ and IL-10 in the culture supernatant were determined by Luminex method. Recipient LEW rats were given infusion of PUVA-SP DC, SP DC or PBS （Control group） through the peripheral vein 7 days before heart transplantation. The survival of cardiac allografts was evaluated by palpation every day. The concentrations of Th1 （IFN-γ, IL-2） and Th2 （IL-10） cytokines in serum of recipients were determined by Luminex method, and the samples of cardiac allograft were collected for pathological evaluation 6 days after transplantation. Results After being co-cultured with donor PUVA-SP, singnificantly more IL-10 and IFN-7 were secreted by recipient DC compared with that by DC cultured with donor SP （P〈0. 01）. Comparing with the infusion of SP DC or PBS, the infusion of PUVA-SP DC significantly decreased the serum levels of IL-2 and IFN-γ in cardiac aliograft recipients, but it increased the serum level of IL-10. The allograft of PUVA-SP DC group survived much longer than those of control group and SP DC group （27. 2d vs 6. 7d and 5. 5d, P〈0.01） with lower rejection grade. Conclusion PUVA-SP DC is a novel negative immunoregulatory tool, which is able to mediate Th2 immune deviation in vivo and down-regulate the reaction of allograft rejection.