青藤碱（SN）增强iPS定向分化为imDC及SN-iPS-imDC诱导小鼠肾脏移植免疫耐受的机制研究

Induction of specific immune tolerance is the best way to achieve long-term survival of the graft. imDC is considered to be a regulatory target for inducing immune tolerance and has become a hot topic of current research. However, the source of imDC is limited and it is easy to mature in the body, which is the bottleneck of its application. iPS cells are widely available and do not involve ethical issues, and sinomenine (SN) can inhibit imDC maturation by regulating the signaling pathways in imDC cells. Therefore, this project is based on our previous research on the differentiation of mouse bone marrow hematopoietic stem cells and induced pluripotent stem cells (iPS) into imDC. We first combining SN with iPS technology, and we intend to use SN to intervene iPS to differentiate into imDC (SN-iPS-imDC), and injecting the donor-derived SN-iPS-imDC into mouse kidney transplantation model. The aim of this study is to clarify: ①the method of SN intervention iPS efficiently differentiated into imDC; ②the mechanism of SN-iPS-imDC remains immature; ③the mechanism of SN-iPS-imDC induced donor-specific immune tolerance in mouse kidney transplantation. Innovative and breakthroughs were proposed for "iPS efficiently differentiated into imDC" , "explore new sources of imDC"and "maintaining the immature state of imDC in vivo ", and explore a new way for SN-iPS-imDC to induce immune tolerance.

诱导特异性免疫耐受是移植器官长期存活的最佳途径。imDC被认为是诱导免疫耐受的调控靶点，成为目前研究的热点。但imDC来源受限和在体内易成熟是其应用的瓶颈。诱导性多潜能干细胞（iPS）来源广泛且不涉及伦理学问题，青藤碱（SN）可通过调控imDC内信号通路抑制其成熟。因此，本项目在我们前期对小鼠骨髓造血干细胞和iPS定向分化为imDC的研究基础之上，首次将SN与iPS技术相结合，拟用SN干预iPS定向分化为imDC（SN-iPS-imDC），将供者的SN-iPS-imDC输入小鼠肾移植模型体内。旨在阐明①SN干预iPS高效分化为imDC的方法；②SN-iPS-imDC保持未成熟状态的机理；③SN-iPS-imDC诱导小鼠肾移植供者特异性免疫耐受的作用和机制。为“解决imDC来源”、“维持imDC在体内的未成熟状态”提出创新和突破，探索SN-iPS-imDC诱导免疫耐受的新途径。

imDCs表面缺乏共刺激分子的表达，是诱导免疫耐受的调控靶点。但其来源有限且在体内易成熟，成为应用瓶颈。iPS来源广泛、不受伦理学限制，有望成为imDCs的新来源。SN可降低imDCs表面共刺激分子的表达和NF-κB的活性，从而抑制imDCs的成熟。本研究用SN作用于iPS定向分化为imDCs的过程，获得能够保持未成熟状态的iSN-iPS-imDCs，通过观察SN-iPS-imDCs在体内、外的作用及机制，为imDCs应用于移植免疫领域奠定实验基础。.本研究建立了标准的SN-iPS-imDCs培养体系并对其单克隆进行鉴定。SN-iPS定向分化的过程分为三个阶段，第一阶细胞出现三胚层结构，第二阶段细胞表面高表达CD45和CD11b，第三阶段获得SN-iPS-imDCs，其低表达CD80、CD86和MHC-Ⅱ，高表达CD11c。LPS处理SN-iPS-imDCs 48 h，SN-iPS-DCs细胞表面CD80、CD86、MHC-Ⅱ阳性率，培养上清液中的IL-2、IL-12、IFN-γ和TNF-α分泌量以及刺激CD4+T细胞增殖的能力显著低于iPS-DCs，而CD4+CD25+Foxp3+Tregs比例、凋亡率及Bax、Fas基因和actived-caspase3、actived-caspase9、actived-PARP蛋白相对表达量显著高于iPS-DCs。在小鼠耳-背皮肤移植模型中，术后各时间点SN-iPS-imDCs组皮肤排斥反应评分显著低于PBS、SN和iPS-imDCs组；移植皮片存活时间与其他组比较显著延长；对供者淋巴细胞的反应性显著低于无关第三者C3H小鼠和iPS-imDC组；移植皮片病理HE染色炎性反应显著低于其它组。本研究首次将中草药单体SN与iPS相结合，成功获得了SN-iPS-imDCs，揭示了SN具有维持iPS-imDCs未成熟的特性；阐明了SN-iPS-imDCs通过诱导CD4+CD25+Tregs、CD4+CD25+Foxp3+Tregs产生、促进IL-10、TGF-β分泌等机制，抑制T淋巴细胞的增殖和活化，且诱导移植免疫低反应性与细胞凋亡有关。此研究探索“SN—iPS细胞—SN-iPS-imDCs—诱导供者特异的免疫低反应性”这一新方法，为SN-iPS-imDCs用于诱导免疫耐受领域提供理论依据，为中医药与现代新技术相结合应用于移植免疫领域开