CD47信号传导位点定位及其在移植物损伤中的作用机制研究

Ubiquitously expressed cell surface glycoprotein-CD47 plays two main roles in transplantation. Firstly, its extracellular domain is able to active the immunosuppression receptor SIRPalpha which locates on macrophages, thus inhibiting allograft rejection of hematopoietic cells caused by phagocytosis. Secondly, CD47 serves as a receptor, which can be activated by TSP1 and transmits intracellular signals that inhibit cell survival, aggravating injury and rejection of solid organ transplants. The mechanisms by which activated CD47 induces multiple intracellular signaling have been intensively studied. However, the specific sites through which CD47 transmits the signals have not been clarified at the molecular level. In addition, the key mechanism of activated CD47 for inducing graft injury still remains unknown. In this project, we plan to combine the protein sequence analysis with the secondary structure prediction for CD47 mutation design. We will introduce replacements or mutations into the transmembrane domains and cytoplasmic domains of CD47 which may play a role in cognate partner binding and signal transduction, while the structural determinants on CD47 that mediate binding to SIRPalpha remain unchanged. Afterwards, we will check the abilities of the CD47 mutants to bind and activate the corresponding proteins, and check the changes in the concentration of the second messenger molecules at the cellular level in the presence of CD47 activator, thereby screening and localizing the sites of CD47 which mediate the transmission of signals. Futhermore, we will explore the mechanism through which activated CD47 promotes graft injury. Our observations will provide a theoretical basis for the treatment of transplant rejection, and extend the current understanding of the signal transfer mechanism of CD47.

遍在化表达的CD47在移植领域有两大热点功能。第一是其胞外结构域能够激活巨噬细胞表面的抑制受体SIRPalpha，抑制吞噬导致的细胞移植排斥。第二是CD47能够被TSP1激活，向细胞内传递生存抑制信号，加剧实体器官的移植损伤与排斥。CD47激活引发的多种信号通路机制已有大量报道，然而其传导下游信号的具体位点至今未被详细阐明。此外，哪种通路是其影响移植物损伤的关键机制也尚不明确。本项目拟运用蛋白质序列分析、结构预测等手段，保持CD47激活SIRPalpha的结构域不变，对CD47分子可能参与通路蛋白结合与信号传递的区段进行替换或突变。而后在CD47激活物作用下，从细胞水平对突变CD47结合、激活相应通路蛋白能力的变化及通路下游各信使分子水平的变化进行分析，从而筛查并定位信号传导位点，探索CD47在移植物损伤中的作用机制。为移植排斥治疗提供理论依据并深化我们对CD47信号传递机制的理解。

遍在性表达的膜蛋白CD47在移植领域有两大相反的热点功能。良性功能为其胞外域能激活巨噬细胞表面的吞噬抑制受体SIRPalpha，抑制吞噬导致的细胞移植排斥。恶性功能为CD47可阻碍细胞应激条件下维生的自噬途径，并结合TSP1，通过穿膜区与胞质区向胞内传递生存抑制信号，加剧实体器官的移植损伤与排斥。因而，深入研究CD47功能区域对移植领域具有重要意义。. 本课题对尚未明确的CD47向胞膜表面定位的分选转运机制及其干扰细胞自噬与传导生存抑制信号的功能位点展开研究，并评价相应CD47突变体的吞噬抵抗能力。在保持CD47良性抗吞噬功能域不变的前提下，对CD47可能参与自身分选转运与信号传递的区段进行替换或突变。而后检测CD47细胞内外分布及亚细胞定位，分析突变对CD47转运的影响；同时在应激条件下，从细胞水平评价突变对CD47生存抑制能力的影响。筛查定位CD47分子功能位点，探索CD47在移植物损伤中的作用机制。建立吞噬体系以评价不影响细胞生存的CD47突变体的抗吞噬能力，以期最终获得不传递生存抑制信号同时能保护细胞免于吞噬的CD47突变体。. 在CD47分选转运机制方面，我们发现CD47第五螺旋与其胞质尾端同时介导CD47向膜表面的定位。数据显示，当仅替换CD47第五螺旋或仅删除胞质尾端时，CD47定位不受突变影响。然而，当同时替换CD47第五螺旋并删除胞质尾端时，CD47严重滞留于细胞内部，这种现象在多种细胞中均显著呈现。随后我们为排除外源螺旋引入可能导致的CD47折叠失误，对螺旋5及胞质尾端同时进行删除，同样发现了CD47的细胞内严重滞留及膜表面定位失败。这表明我们可能发现了CD47参与自身转运的关键功能区。. 在CD47生存抑制功能方面，我们发现辐照压力下，与野生型CD47相比，CD47胞内区突变能够特异性保护细胞活性并保护细胞自噬能力。同时，与其他报道相同，我们发现使用GPI锚定区替换CD47穿膜区和胞内区可以阻断其生存抑制信号传导，我们利用人外周血单核细胞建立了体外吞噬体系用以评价这个不传导生存抑制信号的突变体能否保护细胞免于吞噬。. 综上，本课题的研究结果为深入阐明CD47分选转运机制提供了线索；为最终获得适用于异种移植的CD47改造分子奠定理论基础；为移植排斥治疗提供备选方案并扩展了我们对CD47信号传导机制的理解。