小鼠小肠移植术前输注表达吲哚胺2，3双加氧酶的供者树突细胞抑制排斥反应

The inhibitory effect of donor-derived DCs highly expressing functional indoleamine 2,3-dioxygenase on acute rejection in mice small bowel transplantation

目的 观察干扰素γ（IFN-γ）诱导供者（C57BL／6小鼠）脾树突细胞（DC）表达吲哚胺2，3双加氧酶（IDO）的情况；研究受者（BalB／c小鼠）小肠移植术前输注高表达IDO的供者DC对排斥反应的抑制作用。方法 用IFN-γ诱导供者DC表达IDO；半定量逆转录聚合酶链反应、免疫印迹法及毛细管电泳法检测IDO表达水平及活性，混合淋巴细胞培养（MLR）测定IDO刺激T细胞增殖的能力。利用小鼠异位小肠移植模型，设单纯移植组、DC输注移植组（术前输注供者脾DC 2×10^6个）及诱导DC输注移植组（术前输注经IFN-γ诱导的供者脾DC 2×10^6个），术后观察移植肠存活时间并行病理学检查。结果 经IFN-γ诱导的脾DC IDO分子mRNA转录水平（相对量）、IDO蛋白表达水平（相对量）及培养液中犬尿氨酸浓度分别为（1．23±0．02）、（2．74±0．01）以及（76．52±0．44）μmol／L，未经IFN-γ诱导的脾DC分别为（1．05±0．05）、（1．40±0．17）及（43．31±0．48）μmol／L，前者显著增强（P〈0．01）。脾DC对同种T细胞增殖的刺激作用在IFN-γ诱导后减弱，在加入IDO的特异性抑制剂后增强。输注诱导DC移植组移植肠中位存活时间（12d）较单纯移植组（6d）及输注DC移植组（7．5d）显著延长（P〈0．01），而移植肠病理分级显著性降低（P〈0．05）。结论 IFN-γ可诱导小鼠脾DC高表达活性的IDO，后者可减弱DC刺激T细胞增殖的能力。受者术前输注高表达IDO的供者DC能够诱导针对供者的特异性免疫耐受而减轻排斥反应。

Objective To investigate the indolearnine 2,3-dioxygenase （IDO） expression of murine splenic dentritic cells （DCs） with or without interferon-γ （IFN-γ） induction and the inhibitory effect of donor-derived DCs highly expressing functional IDO on acute rejection in mice small bowel transplantation （SBT）. Methods DCs were isolated from murine spleen and cultured. The phenotypes of DCs were determined by flow cytometry. The IDO expression level and activity of DCs with or without IFN-γ induction were investigated by SQRT-PCR, Western blot and capillary electrophoresis. The alterations in stimulating ability to allogenic T lymphocyte proliferation through DCs with/without IFN-γ induction were observed by single mixed lymphocyte culture. Heterotopic SBT models were established （C57BL/6→BalB/c） and the following groups were set up, including group without treatment,group treated with induced DCs [donor-derived splenic DCs （2 ×10^6） induced by IFN-γ were preoperatively intravenously injected to recipient] and group treated with DCs [donor-derived splenic DCs （2× 10^6） without IFN-γ induction were preoperatively intravenously injected to recipient]. The survivals of grafts were observed. On the post-operation day （POD） 6, grafts were individually collected for pathological examination. Results The expression level of IDO mRNA and protein in murine splenic DCs as well as the kynurenine concentration of splenic DCs supernatants （ 1.05 ± 0. 05,1.40 ± 0. 17 and 43. 31 ± 0. 48μmol/L respectively） were correspondingly increased by IFN-γ induction （ 1.23 ± 0. 02,2. 74 ±0. 01 and 76. 52 ± 0. 44μmol/L respectively）. The stimulating ability to allogenic T lymphocyte proliferation of splenic DCs was significantly was reduced with IFN-γ induction but enhanced by application of specific inhibitor 1-methyl-tryptophan （1-MT）. The graft survival in the group treated with induced DCs （median 12 days） was significantly longer （P〈0. 01） than that in the group treated with DCs （7. 5 days） and the group without treatment （6 days）. The pathological grades in the group treated with induced DCs （subtle） were significantly lowered as compared with those in the group treated with DCs （moderate） and the group without treatment （severe） （P〈0.05）. Conclusions Murine splenic DCs had the ability of functional IDO expression. The high expression of functional IDO from splenic DCs was due to IFN-γ induction and made stimulating ability of splenic DC to allogenic T cells proliferation lower. Pre-operative medication with donor-derived DCs highly expressing functional IDO may inhibit rejection in mice SBT.