中药天花粉蛋白衍生肽通过Treg抑制同种异体肺移植慢性排斥的应用基础研究

Transplantation is a sole approach for effective treatment of lung failure. However, the chronic rejection of grafts with obliterative bronchiolitis (OB) prevents the recipients and their grafts from long survival. The treatment of recipients with immuno- suppressants however has limited effects and at high risk for combining infections and carcinogenesis. Trichosantin (Tk) is a plant protein isolated from a Chinese medicinal herb. We have reported that Tk-P, a Tk-derived peptide, was able to obviously prolong the survival of mouse cardiac allo-grafts by activating and expanding CD8(+)CD28(-) Treg and nTreg subsets, which showed potent capability of repressing the Th1-dominated immune responses for the rejection. In this connection, this study aims: (1) To explore whether Tk-P is also active in suppression of rejection for mouse lung allo-grafting by diminishing OB development. (2) To figue out a particular atlas with regard to the type and working range of the Tregs involved and their association with prognosis. (3) To elucidate the role of the Tk-P-activated Tregs in tolerance induction of the lung allo-grafting. (4) To study molecular mechanisms and signaling pathways included in the Tk-P-related negative regulation by sequencing of essential genes, bio- informational analysis and by a series of in vitro and in vivo experiments. In this way, the Tk-derived new drugs with high efficiency and low toxicity would be developed to lay a sound background for their applications in clinical organ transplantation.

肺移植是治疗衰竭性肺部疾病唯一有效的方法，然而术后慢性排斥（闭塞性细支气管炎）是限制受者长期存活的关键因素，免疫抑制剂治疗效果不佳，并会导致感染及肿瘤高发。我们首次报道，中药天花粉蛋白衍生肽 (Tk-P) 能显著延长Th1主宰的小鼠同种异体移植心脏存活期，并与其选择性诱导CD8(＋)CD28(－)Treg及nTreg有关。据此，本项目拟探讨和确认Tk-P可通过诱导特定类型Treg抑制小鼠肺异体移植排斥反应和延缓OB的发生发展，绘制中国人群肺移植受者Treg类型和作用的特征性图谱及其与预后的关联性，了解Tk-P诱导Treg在肺移植免疫耐受中的作用，并通过基因测序、生物信息学和一系列体内外实验揭示Tk-P实施负向调节的分子免疫学机制和关键信号通路，以发展高效低毒的原创新型药物，为临床器官移植提供新的制剂和手段奠定基础。

1..从临床前药物学角度评价了天花粉蛋白衍生肽Tk-PQ治疗同种异体肺移植慢性排斥体内外的安全性，Tk-PQ用药对正常小鼠/大鼠以及同种异体移植鼠均无毒副作用，肝脏、肾脏和脾脏病理结果显示其对鼠无任何不良影响。.2..给予同种异体肺移植动物模型Tk-PQ 注射显示其对同种异体肺移植慢性排斥反应具有良好的治疗效果。治疗组小鼠移植气管保持了正常的组织结构，未出现严重的闭塞情况，未见异常肉芽肿形成；同时发现，Tk-PQ可抑制气管上皮细胞的丢失，保护上皮组织免于炎症损伤，维持了上皮细胞及粘膜下腺体组织的正常功能，最为明显的是显著降低OB小鼠气管纤维化蛋白α-SMA的表达，表明Tk-PQ能有效抑制OB发生发展中的组织纤维化。.3..从2个角度揭示了TK-PQ延缓OB发生发展的细胞及分子免疫学机制。（1）通过诱导受体免疫格局Th1向Th2/Treg免疫应答偏移，Th2/nTreg细胞比例增加，特异性转录因子GATA3+/Foxp3+和IL-4/IL10/IL33细胞因子水平显著高表达，抑制局部炎症反应，通过生信分析Th2/nTreg分化可能涉及usp7和IRF4；（2）Tk-PQ 明显抑制效应活化T淋巴细胞增殖, 并从基因和蛋白水平确认下调PI3K-Akt信号通路关键基因是Tk-PQ发挥抑制的分子机制。.4..建立了中国健康人群外周血CD4+Foxp3+Treg细胞的正常值范围，以此为对照我们分析了调节性T细胞在中国部分人群肺移植中的谱系特点、移植后的变化规律，发现CD4+Foxp3+Treg细胞的时间点变化在长期预后良好组、术后存活组及术后死亡组存在差异。长期预后良好组术前术后CD4+ CD25+Foxp3+ Treg比例均较高，术后死亡组患者CD4+ CD25+Foxp3+ Treg比例偏低，说明肺移植患者外周血中CD4+ CD25+Foxp3+ Treg比例可作为移植预后的细胞学生物标记物，提示诱导Treg细胞比例增加可以预防移植术后患者死亡，增加患者长期生存率。.5..初步揭示中国部分人群肺移植患者移植前后外周血43种细胞因子的变化规律，发现IL-10、IL-10/IFN-γ、IL-6、IL-8、TNF-α、G-CSF、GM-CSF在预测肺移植预后中具有重要的临床价值。.6..项目资助下获得授权专利2项，标注基金号发表 18篇论文，培养1名博士后，4名博士研究生。