乏氧环境中肿瘤细胞释放的组织因子微粒（TFMPs）通过激活凝血系统促进肺癌复发转移的机制研究

Recurrence and metastasis of lung cancer are the main obstacle to the survival of patients. However, the molecular mechanism underlying circulating tumor cells forming recurrence and metastasis after radical surgery has not been elucidated. Recent study has demonstrated that the release of tissue factor-bearing microparticles (TFMPs) could be induced by hypoxia. And TFMPs played a key role in the activation of coagulation system. Furthermore, the activation of coagulation system was related to more aggressive tumor behaviors. Our previous work found that in some patients after lung cancer radical surgery the activated partial thromboplastin time was significantly shortened, and D-dimer, fibrinogen as well as platelets were significantly increased. Moreover, inhibition of TF expression in lung cancer cells could reduce the growth of tumor. And there was relationship between the increased activity of TFMPs after radical surgery and poor prognosis.To this end, we propose the hypothesis: Hypoxia induces circulating tumor cells to release TFMPs, which promotes recurrence and metastasis of lung cancer through activating the coagulation system. To test this hypothesis, we will carry out hypoxia environment, TFMPs quantity and activity assays, coagulation assays, gene knockout, plasmid as well as animal experiments to reveal that interaction of hypoxia and TFMPs activates the coagutlation system followed by recurrence and metastasis of lung cancer, which will provide new targets for the prevention and treatment of lung cancer with recurrence and metastasis.

肺癌复发转移是制约肺癌患者生存的主要瓶颈，但肺癌根治术后循环肿瘤细胞如何形成复发转移灶的机制尚未阐明。研究发现TFMPs的释放能够被乏氧环境诱导，且其在体内凝血系统激活过程中起到关键作用。而体内凝血系统的激活与更加侵袭性的肿瘤行为相关。我们前期研究发现，肺癌术后部分患者活化的部分凝血活酶时间明显缩短，D-二聚体、纤维蛋白原和血小板明显升高；抑制肺癌细胞的TF表达，能抑制肺癌的生长；而且术后肺癌患者循环TFMPs活性的增加与预后差相关。为此我们提出假说：乏氧环境可诱导循环肿瘤细胞释放TFMPs，通过激活体内凝血系统促进肺癌的复发转移灶的形成。为验证此假说，我们使用乏氧干预、TFMPs数量与活性检测、凝血相关指标检测、基因敲除、质粒构建以及裸鼠体内肺癌转移瘤模型等手段，阐明乏氧与TFMPs的相互作用进而激活凝血系统，促进肺癌复发转移的具体分子机制，为预防和治疗肺癌复发转移提供新的靶点。

一、.项目背景. 目前肺癌的 5 年总生 存率仍仅有 18%，其中绝大多数的肺癌相关性死亡是由于肿瘤出现了能抵 抗多种治疗的复发和转移。目前肺癌出现复发转移的相关机制仍未完全阐明，发现新的治疗和预防肺癌复发转移的方法已经成为肺癌治疗上的巨大挑战。.既往研究中，TFMPs 是肺癌肿块完整切除后循环中的主要促凝血因子，具 有促进肺癌增殖、迁移、侵袭等作用。TFMPs 的释放既能够被乏氧环境诱导，也能促进乏氧微环境形成。本项目中，我们推测肺癌的乏氧微环境通过激活 ERK/ EGR-1 通路刺激了肿瘤细胞释放 TFMPs，TFMPs 进一步激活凝血系统，促进肺癌生长和转移。.二、.主要研究内容. 组织因子微粒（ TFMPs）可通过乏氧环境激活ERK/ EGR-1通路，促进肺癌的转移。.三、.重要结果、关键数据和科学意义. 本研究明确了乏氧环境可促进A549肺癌细胞迁移，对肺癌细胞增殖无明显影响，且乏氧环境可促进TF的表达和TFMPs的分泌和活性增加。进一步通过siRNA沉默A549肺癌细胞的乏氧诱导因子HIF1α，也发现了可促进肺癌细胞迁移，TF表达上升，TFMPs的分泌和活性增加。通过Western blot分析，乏氧对肺癌细胞的影响主要通过Erk1/ 2和Egr-1通路。本研究对治疗肺癌转移提供了新的思路，具有一定的应用前景。