基于急性抗体介导排斥反应的NTPDase1代谢胞外ADP失衡、激活B细胞及其损伤的机制研究

Our project supported by the National Natural Science Foundation of China in driblet in 2012 and found that the unbalance with extracellular ADP metabolized by nucleoside triphosphate diphosphohydrolases 1 (NTPDase1) and then platelet activated is one of important steps to damage the transplanted organ in acute antibody-mediated rejection (AMR). Moreover, the B cell proliferation was inhibited while the unbalance was corrected by NTPDase1 pretreatment. But the mechanism of the B cell and transplanted organ function affected by extracellular ADP metabolized by NTPDase1 in acute AMR is not clear. In our study, NTPDase1 transgenosis nude mice, NTPDase1 gene knock-out nude mice and wild type nude mice skin grafting model respectively will be induced acute AMR. There are different NTPDase1 expression levels in three kinds of nude mice. The pathological change, ultrastructure and apoptosis of grafting skin in different nude mice will be observed. The proliferation, surface markers expression and antibody formation abilities of B cell in different nude mice will be compared. The ADP receptor of B cell surface and the calcium ion concentration, enzymatic activity, cAMP content in the intra-cellular of B cell in different nude mice will be studyed. The relationship between the metabolism of extracellular ADP by NTPDase1 and the B cell function in acute AMR will be discussed. The mechanism and effect of B cell activation and then its damage to the transplanted skin caused by extracellular ADP metabolized unbalance will be analysed at molecules and histology level. It will open new method to treat acute AMR in transplantation field.

本组2012年国自然面上小额立项课题发现：急性抗体介导排斥反应（Antibody-mediated rejection，AMR）中三磷酸核苷双磷酸水解酶（NTPDase1）代谢胞外ADP失衡可激活血小板、损伤移植器官；NTPDase1预处置校正失衡的同时可抑制B细胞增殖，但NTPDase1改变B细胞功能及其对移植器官影响的机制尚不明。本研究拟分别在NTPDase1表达水平不同的NTPDase1转基因增强表达、基因敲除和野生型裸鼠皮肤移植模型上诱导急性AMR，比较移植皮肤细胞的超微结构和凋亡程度，比较B细胞增殖、表面标志物表达和产生抗体的能力，比较B细胞表面ADP受体和胞内钙离子浓度、酶活性、cAMP生成量的改变，从组织学和分子水平探讨急性AMR中NTPDase1代谢胞外ADP、及其直接作用于B细胞表面和胞内信号传导通路激活B细胞、继而损伤移植皮肤细胞的效应和机制，开辟急性AMR治疗新途径。

项目背景：探讨急性抗体介导排斥反应（Antibody-mediated rejection, AMR）中，NTPDase1降解胞外ADP、及其激活B细胞、活化血小板和对移植皮肤损伤的效应及机制。主要研究内容：分别在NTPDase1表达水平不同的NTPDase1转基因增强表达、基因敲除和野生型裸鼠皮肤移植模型上诱导急性AMR，使用荧光素/荧光素酶法、实时荧光定量PCR法、HPLC法、免疫荧光法、流式细胞、免疫组化、电镜等方法，从组织学和分子水平研究急性AMR发生时B细胞NTPDase1的表达活性、移植皮肤NTPDase1 mRNA表达量、胞外ADP浓度、B细胞体积和表面抗原表达量、血小板移动平均速率，以及移植皮肤细胞的超微结构和细胞凋亡；研究不同剂量的外源性NTPDase1预处置对急性AMR发生时血小板活化和移植皮肤损伤的效应。重要结果和关键数据：急性AMR中，NTPDase1的表达水平越高，裸鼠胞外ADP的浓度越低，B细胞体积和表面MHCⅡ型抗原表达量增加的幅度越小，NTPDase1的表达量与胞外ADP浓度和B细胞的活化程度负相关；NTPDase1的表达水平越高，血小板移动的平均速率越小，移植皮肤的损伤程度越轻，NTPDase1的表达量与血小板的活化和移植皮肤的损伤程度负相关；高剂量外源性NTPDase1预处置可以有效地抑制血小板活化、保护移植皮肤，但是高剂量的外源性NTPDase1预处置导致受鼠的出血时间延长51.4%、凝血时间延长44.1%。研究结论及科学意义：NTPDase1降解胞外ADP失衡可能促进了B细胞的活化，并激活血小板、加重裸鼠移植皮肤的损伤，高剂量外源性NTPDase1预处置可以有效地保护裸鼠移植皮肤，但增加出血风险。该课题从组织学和分子水平探讨急性AMR中NTPDase1代谢胞外ADP失衡、直接作用于B细胞表面和胞内信号传导通路激活B细胞、继而损伤移植皮肤细胞的效应和机制，开辟急性AMR治疗的新途径。