利用凝血相關小鼠模型探討胰臟腫瘤微囊胞誘導血栓的角色

Abstract

癌症相關血栓(Cancer-associated thrombosis, CAT)為癌症病人的致死因素之一，發生率僅次於因癌症惡化而死亡，而胰臟癌發生CAT的機率相當高，因此探討胰臟癌相關靜脈血栓形成機制相當重要。先前研究指出，胰臟癌可能藉由癌細胞釋出的微囊胞(Microvesicles, MVs)上表現人類組織因子(Human tissue factor, TF)導致第七因子-組織因子複合體(TF-FVIIa complex)形成，造成凝血外在途徑活化產生血栓。臨床研究顯示癌症病患凝血內在途徑和血小板活化及溫韋伯氏因子(von Willebrand factor, VWF)增加，但其對於MVs誘導癌症血栓的影響機制未明。本研究使用低組織因子表現之胰臟癌細胞株MIAPACA 2(TF-)及過量表達TF的MIAPACA 2細胞株(TF+)，以靜脈血栓模型的方式比較各細胞株所產生的MVs影響第八因子剔除、第九因子剔除、VWF點突變及TXA2合成酶剔除小鼠血栓表現的差異，以此研究腫瘤微粒誘導的血栓的機制。另以免疫缺陷的小鼠原位腫瘤模型研究內生性MVs對於胰臟癌血栓的影響。先前已建立定性、定量MVs及其TF活性的測定方法。在靜脈血栓模型中顯示TF+ MVs會促進野生型小鼠的血栓形成，但在第八因子缺損、第九因子缺損、TXA2合成酶剔除的小鼠MVs所誘導的血栓形成顯著下降，而在VWF點突變的組別沒有顯著差異； TF- MVs在野生型及各基因缺損的小鼠靜脈血栓模型則無促進血栓產生。比較肺栓塞嚴重程度及血栓指標表現推測第九因子對血栓形成的影響大於第八因子。腫瘤小鼠模型發現TF+ 腫瘤在野生型及第八因子缺損小鼠相較TF-腫瘤有較高的腫瘤重量及血栓指標，但在第八因子缺損下腫瘤大小及血栓指標差距顯著縮小。由上述結果推測TF是胰臟癌MVs誘導血栓形成中的關鍵角色，同時也會促進腫瘤發展；而凝血內在途徑因子則會影響MV誘導的靜脈血栓形成，且FIX的影響大於FVIII。血小板功能性亦會影響MVs誘導靜脈血栓形成，但VWF與血小板間之交互作用則較不具顯著影響力。

Cancer-associated thrombosis (CAT) is one of the leading causes of death in cancer patients, preceded only by the death of cancer development. Pancreatic cancer is the common cancer that develops CAT. Thus, it is important to investigate the mechanism of pancreatic cancer-associated thrombosis. Previous researches have shown that Tissue factor (TF) expressed by microvesicles (MVs) released from pancreatic cancer cells formed TF-FVII complex to activate the extrinsic coagulation pathway and induced thrombosis. At the same time, clinical evidence showed that the intrinsic pathway, platelet activation, and VWF increase in cancer patients, but the mechanism of these factors to affect MVs-induced thrombosis remained unclear. We utilize low TF expression MIAPACA 2 original cell and TF overexpressed cell line to investigate the effect of FVIII, FIX, VWF-platelet binding and TXA2 to MVs-induced thrombosis utilizing venous thrombosis model with factor-deficient mice. Furthermore, we used a tumor model with hemophilia A immuno-deficient mice to observe the effect of MVs to pancreatic cancer thrombosis. As the result, we set up quantitative and quantitative analysis method of MVs, and the activity assay of MV-TF expression. We observed significantly higher clotting tendency in WT mice venous thrombosis model injected TF+ MV compared to FVIII-deficient, FIX-deficient, and TXA2-ligand-deficient mice, and the VWF point mutation mice didn’t show the same significance. However, there were barely no clot formation in both WT and factor-deficient mice injected TF- MV. Compare the severity of pulmonary embolism (PE) and the expression of thrombosis marker thrombin-antithrombin complex, we presume that FIX is more important than FVIII in clot formation. In tumor mice model, mice bearing with TF+ tumor have larger tumor and higher thrombosis marker expression compared to those bearing with TF- tumor, but the significant gap of the tumor weight was unobserved under FVIII deficiency. To summarize our results, TF might be the key factor in pancreatic tumor MVs-induced thrombosis, and it promotes tumor growth, too. On the other hand, FVIII, FIX may affect MVs-induced venous thrombosis, and FIX has more important role than FVIII. Furthermore, the platelet functions might affect MVs-induced venous thrombosis, too; however, the binding between platelets and VWF has no significant effect.