负向免疫调节树突状细胞对心脏移植大鼠免疫状态的影响

目的 探讨经体外光化学法(PUVA)处理的供者脾淋巴细胞与受者树突状细胞(DC)共培养后,对移植受者体液免疫、细胞免疫及移植物排斥反应的影响.方法 以DA大鼠为供者,LEW大鼠为受者,建立大鼠腹部异位心脏移植模型.分离供者脾淋巴细胞(SP),制备经PUVA处理的供者脾淋巴细胞(PUVA-SP).在体外分别将供者PUVA-SP和SP与受者骨髓来源的未成熟DC共培养,得到PUVA-SP-DC及SP-DC,流式细胞仪检测上述DC表型.根据受者心脏移植术前1周静脉输注成分的不同,将受者随机分为3组:(1)对照组(n=7):单纯输注磷酸盐缓冲液(PBS);(2)SP-DC组(n=8):输注Sp-DC 5×106个;(3)PUVA-SP-DC组(n=8):输注PUVA-SP-DC 5×106个.每日观察各组移植心的存活状况.移植后第6天,检测受者血清中抗供者特异性IgG水平;通过混合淋巴细胞反应(MLR)检测受者脾脏T淋巴细胞对供者抗原刺激的增殖反应;比较各组受者脾脏体积的大小.结果 供者脾淋巴细胞经PUVA处理后细胞凋亡率为81.93%.正常LEW大鼠DC共刺激分子CD80和CD86阳性率分别为(3.5±0.27)%和(13.0±0.58)%,受者DC与供者SP混合培养后,其CD80和CD86的表达水平为(16.6±0.72)%和(36.5±0.87)%,后者明显高于前者(P<0.01);受者DC与供者PUVA-SP混合培养后,其CD86和CD80的表达率分别为(3.9±0.12)%和(13.4±0.59)%,与正常LEW大鼠DC相当(P>0.05).PUVA-SP-DC组的受者抗供者特异性IgG水平明显低于SP-DC组及对照组(P<0.01).PUVA-SP-DC组受者T淋巴细胞对供者抗原的刺激反应指数为1.66±0.29,明显低于SP-DC及对照组(7.28±0.38、4.19±0.16,P<0.01);而其对无关供者抗原的刺激反应指数为4.37±0.11,与SP-DC及对照组相当(4.51±0.40、4.36±0.14,P>0.05).PUVA-SP-DC组的移植心存活时间比其他两组明显延长(P<0.01),而且其脾脏体积最小.结论 PUVA-SP-DC能够特异性的下调移植受者对供者抗原的细胞免疫及体液免疫反应,从而明显延长移植物存活时间.

Effect of a novel negative immunoregulatory DC on immune status of cardiac allograft recipient rats

Objective To investigate the effect of recipient derived dendritic ceils (DC) loaded with PUVA-treated donor splenic lymphocytes (PUVA-SP) on humoral and cellular immunity and allograft rejection in allograft recipients. Methods Heterotopic vascularized heart transplantation model was established from inbred DA to LEW rat. DA rat spleen lyrnphoeytes were treated with 8-methoxypsoralen plus UVA irradiation (PUVA). LEW rat bone marrow-derived DC were co-cultured with PUVA-treated or untreated DA spleen lymphocytes (PUVA-SP or SP), and the costimulatory molecules (CD86 and CD80) of the treated DC (PUVA-SP-DC or SP-DC) and untreated DC were detected by flow cytometry. Recipient LEW rats were subjected to infusion of PUVA-SP-DC, SP-DC or PBS through the peripheral vein at the 7th day before heart transplantation. Cardiac allgrafts survival was evaluated by daily palpation. Circulating alloantibody IgG levels of recipient rats were determined by flow cytometry, and proliferative responses of recipient T cells to donor or third party antigen were examined by mixed leukocyte reaction (MLR). The spleen was collected at the 6th day after transplantation. Results PUVA treatment effectively induced apoptosis of DA rat spleen lymphocytes. After co-cultured with donor PUVA-SP, recipient DC still maintained an immature phenotype with low levels of CD80 and CD86. Injection of PUVA-SP-DC significantly decreased serum anti-donor alloantibody levels of recipient rats, induced donor antigen specific hyporesponsiveness of recipient T cells,and prolonged allograft survival. Moreover, the spleens of the recipient rats, infused with PUVA-SP-DC,were the smallest compared to those of the recipients infused with PBS or SP-DC.Conclusion PUVA-SP-DC could effectively down-regulate anti-donor cellular and humorat adaptive immunity, leading to prolongation of cardiac allograft survival