ADAM 10在胰腺癌诱发深静脉血栓形成中的研究

Tumor-derived deep venous thrombosis(DVT) is the most important complication after the primary malignancy which is directly threatening the patient’s live and severely reducing the life quality, but the mechanism is inconclusive. At present, there are a few studies on the formation mechanism of tumor-derived thrombosis and no clear conclusion yet. Based on our retrospective clinical study, we found that there are 21.5% maligant tumor patients in 454 DVT patients and compared with other neoplasm, adenocarcinoma cancer patients are more prone to present thrombotic events. Besides, for the idiopathic deep vein thrombosis patients, the MUC-1 realeated CA15-3 antigen is dramaticlly elevated which has the highest sensitivity(76.9%), accuracy(87.1%) and negative predictive value(96%) to detect the malignancy..There is considerable literature support ADAM 10 plays a key role in tumor cell proliferation, metastasis, adhesion, and angiogenesis, and activation of platelet aggregation of thrombus formation. According to our pre-experiments, we demonstrated that down-regulation of ADAM10 will decrease the MUC-1 and other coagulation factors expression. Therefore, we hypothesized that in pancreatic carcinoma patient, ADAM10 promotes hypercoagulation status by regulating cell surface MUC-1 which leads to MUC-1 released into the blood vessels and aggregate blood clotting factors combined to form DVT. We use the nanoparticles its own advantages and characteristics of target binding and we combine the NPs labelling technique, PET-CT imaging and cellular and molecular biology techniques together to verify the proposed ADAM 10 regulates MUC-1 to make pancreatic carcinoma derived thrombosis regulatory pathways in molecular, cellular, tissue, organ and individual each aspects. We believe our view is novl and this research would provide prophylaxis and treatment significance for tumor-derived DVT in the future.

肿瘤极易诱发深静脉血栓形成（DVT），但机制尚无定论。我们对454例不明原因DVT患者筛查隐性肿瘤，发现21.5%的DVT患者为原发性肿瘤病例，并首次报道这些患者中MUC-1相关抗原CA15-3显著升高，阳性率达87.1%。近年有文献报道去整合素-金属蛋白酶10（ADAM10）在肿瘤细胞的增殖，转移，粘附和血管生成中起关键作用，并可通过多种途径激活血小板形成血栓。我们的前期实验已证实下调ADAM10表达后可伴有MUC-1和相关凝血因子的表达下降。因此我们推测：肿瘤患者体内ADAM10可能通过调节肿瘤细胞表面的MUC-1来促进凝血，导致DVT，但是机制需要进一步探明，这一观点目前尚无文献报道。我们利用纳米微粒示踪技术，PET-CT显像技术和细胞分子生物学技术相结合，分别从个体，组织，细胞等多个层面探究ADAM10在胰腺癌诱发DVT中的促凝血机制，为预防肿瘤源性DVT和抑制肿瘤转移提供新思路。

本项目名称为“ADAM 10在胰腺癌诱发深静脉血栓形成中的研究”（项目批准号：81760093，起止年月：2018年01月至2021年12月）。旨在验证ADAM10可通调节肿瘤细胞表面的MUC-1分子表达来激活相应的凝血途径并促进血栓形成，通过体外实验和动物实验在不同层面共同探索一条新的肿瘤源性静脉血栓形成通路。.本项目总体执行情况良好，基本按照原计划顺利完成预定实验内容，成功开展以下研究工作：（1）通过siRNA干扰和质粒转染的方法分别降低和升高ADAM10及MUC-1在胰腺癌细胞中的表达，明确目标凝血相关因子TF，PSGL-1和Cathepsin G在mRNA及蛋白水平表达均受到ADAM10和MUC-1的调控。（2）成功建立裸鼠胰腺癌肺转移的动物模型，在动物实验中验证了ADAM10过表达可以增加转移瘤的数量和大小，同时与对照组相比，在转移灶附近可以观察到明显血栓形成。（3）完成了聚乙二醇SPIO脂质体构建的预实验研究，实现了将磁性纳米颗粒与阳离子聚合物聚乙烯亚胺共轭，然后将聚乙二醇与聚乙烯亚胺共轭以改善纳米颗粒的生物相容性，成功合成附载有ADAM10 siRNA的PEG-PEI-Fe3O4纳米颗粒，并通过细胞毒性实验证实该纳米粒子具有较强的生物毒性，不适于用作活体PET-CT示踪成像。（4）通过生信分析、文献查阅和计算机模拟蛋白质互作的方法深入开展ADAM10对MUC-1作用的调控机制探索，筛选出膜连蛋白A1（ANXA1）可能作为ADAM10的互作蛋白调控MUC-1的表达；同时通过体外细胞实验证实ADAM10的改变可以引起NOTCH-1和PD-L1分子的表达变化，推测ADAM10还可能参与肿瘤微环境及免疫抑制的过程。本项目结题后将在上述研究结果的基础上，继续围绕ANXA1和PD-L1有关内容进一步深入开展肿瘤诱发血栓形成的机制研究。.2020年由于受到新冠肺炎疫情影响，武汉实验室封闭且主要研究人员未能到岗，导致改进纳米材料的探索性工作未能如期开展，但是课题组各位成员均秉承认真负责的态度通力合作，努力克服各种困难积极推进研究工作。本项目严格按照国家自然科学基金委要求，各项预算资金执行良好。在项目实施过程中培养硕士研究生2名，目前已发表SCI论文1篇，完成英文论文撰写2篇并处于同行评审阶段。