受体tolDC联合供体骨髓移植诱导异体复合组织移植免疫耐受的新方法及其机制

Physical reconstruction and functional restoration of human limbs, especially upper limbs, still remains a major challenge in clinical practice. Vascularized composite tissue allotransplantation (VCA) is generally regarded as one of the most promising methods to resolve this challenge. However, the clinical application of VCA is usually frustrated by chronic rejection. The latest research has demonstrated that the infusion of donor bone marrow along with VCA could induce chimerism formation and a certain level of immune tolerance. However, its chimerism status is unstable and accompanied by the risk of graft versus host disease (GVHD), which is related to the existence of mature B, T and NK cell in bone marrow according to our preliminary experiment. Tolerogenic dendritic cell (tolDC) could depress rejection and induce tolerance in rodent heart allotransplantation models. But research of tolDC in VCA tolerance induction is rarely seen. In this study, we will combine the recipient tolDC and purified donor bone marrow in a swine VCA model and observe the survival time and rejection grade of VCA allograft. The in vitro interaction between tolDC and donor T cells or bone marrow cells will be investigated. The regulation effect of tolDC towards Treg/Th17 axis in vivo as well as the corresponding cytokine and transcription factor change will be studied. Role of CD45RB and LFA-1 in the interaction between tolDC, Treg and Th17 will be discussed, too. The ultimate goal of this study is to further understand the molecular mechanism of immune tolerance after VCA and seek for the best immune tolerance induction methods which are applicable for clinical VCA.

人类肢体尤其上肢缺失重建仍是一临床难题，带血管异体复合组织移植（VCA）是目前最佳治疗方案，但常因急慢性免疫排斥而失败。近年国外研究联合应用供体骨髓移植可降低VCA免疫排斥，但嵌合体能否形成尚无定论，且存在移植物抗宿主反应风险。我们预实验也未诱导出嵌合体，可能与移植骨髓中存在成熟B、T、NK细胞有关。致耐受性树突状细胞（tolDC）在小鼠心脏移植中可诱导免疫耐受，但在VCA中作用未知。本课题拟在猪VCA模型中联合应用自体tolDC和去除成熟淋巴细胞的供体骨髓移植，检测嵌合体形成和免疫耐受效果；阐明tolDC表达CD45RB、LFA-1分子对Treg/Th17轴的调控机制及相应细胞因子、转录因子T-bet、RORrt、Foxp3等表达水平变化；分析tolDC与嵌合体形成、免疫耐受或排斥之间的关系，从而揭示VCA免疫耐受形成的分子机制，筛选出最佳诱导方案，为临床VCA免疫耐受提供新的策略。

人类肢体尤其上肢缺失重建仍是一临床难题，带血管异体复合组织移植（VCA）是目前最佳治疗方案，但常因急慢性免疫排斥而失败。近年国外研究联合应用供体骨髓移植可降低VCA免疫排斥，但嵌合体能否形成尚无定论，且存在移植物抗宿主反应风险。我们预实验也未诱导出嵌合体，可能与移植骨髓中存在成熟B、T、NK细胞有关。致耐受性树突状细胞（tolDC）在小鼠心脏移植中可诱导免疫耐受，但在VCA中作用未知。首先，本研究建立了猪的VCA动物模型，通过对受体猪进行全身射线照射、激素和免疫抑制剂的干预处理后，术后30天可在受体内检测到供体嵌合体。同时，在移植后150天异体皮肤复合移植物中，其胫骨干骺端的骨髓仍能保持正常形态。将供体骨髓联合免疫抑制剂应用于异体猪VCA模型中，通过观察术后移植物的大体形态、移植物活检HE染色及扫描电镜，结果表明免疫抑制剂FK506联合供体骨髓移植可延长猪VCA移植物存活时间并降低免疫排斥反应。为了进一步探究tolDC在移植免疫耐受中的作用，及其与骨髓间充质干细胞相互作用的机理，课题组分析了成熟树突状细胞和未成熟树突状细胞外泌体中长链非编码RNA的差异表达分析；通过高通量测序检测了C57小鼠骨髓树突状细胞来源的外体中LncRNAs的表达谱，寻找与免疫调节功能相关的潜在分子靶点。最后课题组对骨髓间充质干细胞源性外泌体对树突状细胞的成熟及免疫耐受的作用及机制进行了研究：将BMSCs和DCs混合培养，对DCs表面标志物表达水平进行检测；结果显示：共培养后DCs的表面共刺激分子分子（CD80、CD86和MHC-II）表达量升高。将DCs与小鼠脾脏的naïve CD4+ T 细胞共培养，通过流式细胞术分析CD4+T细胞增殖情况和CD4+CD25+Foxp3+T（致耐受性T细胞）细胞占比，结果显示B-exo处理过的DCs可抑制CD4+T细胞的增殖并诱导CD4+CD25+Foxp3+T细胞的分化。综上所述，本项目对诱导VCA免疫耐受形成的分子机制进行了深入研究，进一步明确了BMSCs源性外泌体诱导tolDCs生成进而促进Treg分化的分子机制，同时筛选出VCA的最佳免疫耐受诱导方案，为临床VCA移植物的长期耐受提供新的策略。