吲哚胺2，3-双加氧酶在间充质干细胞诱导肾移植免疫耐受中的作用

摘要

目的研究探讨骨髓间充质干细胞（MSC）是否能够诱导肾脏移植物免疫耐受的产生以及吲哚胺2，3-双加氧酶（IDO）在MSC介导免疫调节反应中的作用。方法在BALB/c小鼠接受C57BL/6小鼠移植肾脏后24小时，C57BL/6小鼠来源的正常（wt）或IDO基因敲除（IDO-/-）的MSC（1×106）经静脉注入到移植受体中，分别作为wt-MSC治疗组和IDO-/--MSC治疗组，每组6只。以6只未注射的BALB/c移植小鼠受体为未治疗组。以移植物排斥反应所致小鼠死亡或术后100天设定为研究终点。长期存活的BALB/c肾移植受体在移植术后100天，接受来自C57BL/6供体或第三方移植物供体C3H（H-2k）小鼠的皮肤移植，监测移植皮肤情况。对3组小鼠进行移植物组织病理学观察，免疫组化评估肾脏组织Foxp3+细胞水平。用流式细胞技术进行抗原特异性抗体和细胞表型检测，用混合淋巴细胞反应（MLR）评估树突细胞（DC）和T细胞功能。结果本研究发现wt-MSC治疗可诱导受体产生同种异体移植物免疫耐受，表现为移植物病理检查结果正常、未发现抗原特异性抗体水平升高、免疫耐受受体中耐受性树突细胞（Tol-DC）数量显著增多等。同时，在免疫耐受的受体脾脏和肾移植物中均可发现大量CD4+CD25+Foxp3+调节性T细胞（Treg）。这些结果均提示Treg在MSC诱导免疫耐受中的重要作用。值得关注的是，经IDO-/--MSC处理的移植受体中，MSC丧失了其诱导同种异体移植物的免疫耐受的能力，肾移植物很快被排斥，同时机体移植物的排斥反应变化与未治疗组相同。结论由MSC分泌的IDO通过促进Treg的生成，在诱导肾脏移植免疫耐受中起着关键性的作用。本研究将为MSC在器官移植中的临床应用提供理论及临床转化依据。%Objective To determine whether mesenchymal stem cells(MSCs)can induce the establishment of kidney allograft tolerance,as well as indoleamine 2,3-dioxygenase(IDO)contributes to the immunoregulatory functions of MSCs in that process. Methods MSCs(1×106)from wild-type(wt-MSCs)or IDO-knockout (IDO-/--MSCs)C57BL/6 mice were injected intravenously into BALB/c recipients 24 hours after receiving a life-supporting orthotopic C57BL/6 renal graft. Recipients treated with either wt-MSCs or IDO-/--MSCs were used as two study groups(n=6/group). In addition,6 native BALB/c mice were used as controls. Samples were collected at endpoint of graft rejection or on postoperative day(POD)100. Long-term surviving BALB/c kidney allograft recipients received full-thickness skin grafts(1 cm×1 cm)from C57BL/6 donor and C3H mouse strains on POD100 and were monitored daily. The graft pathology,immunohistochemistry,flow cytometry,mixed lymphocyte reaction were used for detecting graft rejection,intragraft Foxp3+ cell infiltration,donor-reactive antibody and cellular phenotypic expression,dendritic cell(DC)and T cell function,respectively. Results wt-MSC-treated recipients achieved allograft tolerance with normal histology and undetectable antidonor antibody levels. Tolerant recipients demonstrated significantly high frequencies of tolerogenic dendritic cells(Tol-DCs). In addition,high frequencies of CD4+CD25+Foxp3+regulatory T-cells(Tregs)were found in recipient spleens and donor grafts,implying the important role of Tregs in the MSC-induced tolerance. Interestingly,renal allograft recipients treated with IDO-/--MSCs, were unable to achieve allograft tolerance,suggesting that functional IDO was necessary for the immunosuppression observed with wt-MSC treatment. Conclusion IDO secreted by MSCs was responsible for induction of kidney allograft tolerance through generation of Tregs. This study supports the clinical application of MSCs in transplantation.