CAJ | 학술논문

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。
목적연구탐토골수간충질간세포（MSC）시부능구유도신장이식물면역내수적산생이급신타알2，3-쌍가양매（IDO）재MSC개도면역조절반응중적작용。방법재BALB/c소서접수C57BL/6소서이식신장후24소시，C57BL/6소서래원적정상（wt）혹IDO기인고제（IDO-/-）적MSC（1×106）경정맥주입도이식수체중，분별작위wt-MSC치료조화IDO-/--MSC치료조，매조6지。이6지미주사적BALB/c이식소서수체위미치료조。이이식물배척반응소치소서사망혹술후100천설정위연구종점。장기존활적BALB/c신이식수체재이식술후100천，접수래자C57BL/6공체혹제삼방이식물공체C3H（H-2k）소서적피부이식，감측이식피부정황。대3조소서진행이식물조직병이학관찰，면역조화평고신장조직Foxp3+세포수평。용류식세포기술진행항원특이성항체화세포표형검측，용혼합림파세포반응（MLR）평고수돌세포（DC）화T세포공능。결과본연구발현wt-MSC치료가유도수체산생동충이체이식물면역내수，표현위이식물병리검사결과정상、미발현항원특이성항체수평승고、면역내수수체중내수성수돌세포（Tol-DC）수량현저증다등。동시，재면역내수적수체비장화신이식물중균가발현대량CD4+CD25+Foxp3+조절성T세포（Treg）。저사결과균제시Treg재MSC유도면역내수중적중요작용。치득관주적시，경IDO-/--MSC처리적이식수체중，MSC상실료기유도동충이체이식물적면역내수적능력，신이식물흔쾌피배척，동시궤체이식물적배척반응변화여미치료조상동。결론유MSC분비적IDO통과촉진Treg적생성，재유도신장이식면역내수중기착관건성적작용。본연구장위MSC재기관이식중적림상응용제공이론급림상전화의거。
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.