以小鼠模型探討腫瘤微粒在癌症相關靜脈血栓之角色

Investigating the role of microvesicles in cancer-associated venous thrombosis by using mouse models

林昭怡(Chao-I Lin)

指導教授 ： 林淑華

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202002651

摘要

癌症病人第二大死因為靜脈栓塞，其發生機率是常人的4-7倍，其中以胰臟癌病人比例最高。文獻指出癌症病人發生靜脈栓塞的機制與腫瘤細胞表現的組織因子(tissue factor, TF)會與血流中的凝血因子結合啟動外在凝血路徑及腫瘤細胞釋放TF+腫瘤微粒(TF+ MV)吸引血小板聚集。目前推測不同腫瘤細胞有各自的特定機制形成栓塞，與腫瘤細胞表面帶有的受體(receptor)或其他膜蛋白不同有關。除了由組織因子誘發外在途徑活化引發的血栓形成外，在癌症病人體內也可發現內在途徑被活化的證據。在癌症病人血漿中可發現血小板或紅血球釋放的TF- MV可透過內在途徑活化凝血系統，可能為組織因子陰性(TF-)腫瘤發生血栓的機轉。本研究使用AsPC-1野生株(TF+)及利用CRISPR技術得組織因子剔除株(TF-)，建立胰臟癌原位注射模型並以A型血友病小鼠探討內在途徑及外在途徑對腫瘤生長、轉移、是否分泌人類腫瘤微粒並誘發血栓方面造成之影響。而為了解腫瘤微粒在癌症靜脈栓塞扮演的角色，目前已建立分離、定性、定量的流程並將其注射至缺乏不同凝血因子的小鼠中觀察組織因子陽性腫瘤微粒(TF+ MV)對不同凝血路徑因子缺乏小鼠造成之影響，包括血栓重量及休克病理原因探討。實驗結果顯示TF主導AsPC-1腫瘤微粒產生血栓，且TF-的腫瘤細胞釋放之腫瘤微粒未見血栓的生成，而血友病小鼠產生的血栓重量較小且具統計差異顯示內在路徑扮演一定角色。腫瘤生長方面，以野生型或A型血友病NSG來看TF+與TF-的腫瘤大小無顯著差異；以TF-腫瘤而言，血友病NSG相比野生型NSG腫瘤較小。以此推斷內在途徑在TF-腫瘤生長扮演角色。目前無法從腫瘤移植小鼠中分離出人類腫瘤微粒，故需要再改良實驗設計及探討AsPC-1細胞造成癌症相關栓塞機制是否主要由腫瘤微粒所誘發。綜合上述結果可推測內在途徑與外在途徑相互作用在腫瘤生長及誘發產生血栓，未來可根據此研究建立之模型及結果探討腫瘤與腫瘤微粒如何產生靜脈栓塞之機制。

關鍵字

癌症靜脈栓塞 ； 組織因子 ； 內在途徑 ； 外在途徑 ； 腫瘤微粒

並列摘要

Cancer patients have a 4 to 7 folds increased risk of venous thromboembolism (VTE) which is the second leading cause of death. Pancreatic cancer patients have relatively higher incidence of VTE than other cancer types. One of the major mechanisms has been proposed that tissue factor (TF) expressed by tumor can bind with FVIIa from blood circulation to activate the extrinsic pathway, and the tumor derived TF positive microvesicles (MV) can recruit platelets to develop thrombosis. In previous studies, different cell lines with different cell surface receptors and membrane proteins can cause cell type specific mechanisms of VTE, thus additional studies still needed to provide the responsible molecular mechanism. Besides, there’re clinical evidences show that intrinsic pathway is activated in cancer patients. However, platelets and erythrocytes derived MV are found to activate coagulation system through the intrinsic pathway can be the possible mechanism to trigger TF negative cancer associated VTE.
We used AsPC-1 cell line (TF+) and its TF knockout clones (TF-) produced by CRISPR/Cas9 system and established the orthotopic injection models in NSG and NSG mice with hemophilia A to investigate how intrinsic and extrinsic pathway affect tumor growth, metastasis and the ability to release human MV.
To clarify the role of MV plays in cancer associated VTE in vivo, we first isolated them shed by AsPC-1 and its TF knockout clones to characterize their size distribution, concentration, morphology, the components on its membrane and injected them separately into mice with different clotting factors deficiency then.
Our results suggest that TF is the leading factor of thrombus formation induced by tumor derived MV and tumor growth. The extrinsic pathway and intrinsic pathway interaction may be included in the mechanisms of tumor growth and cancer associated VTE. TF+ MV caused most of the mice to have clot in the IVC while MV derived from TF negative cell cannot induce that. Thrombi in mice with hemophilia are smaller than their littermate control which indicates that intrinsic pathway participates in the process. In tumor growth, TF+ tumor grew bigger than TF- tumor in NSG mice. On the other hand, TF- tumor grew in NSG mice bigger than mice with hemophilia. Nevertheless, we haven’t isolated the human tumor derived MV successfully.
On the base of this study, we can use the established models to know more about how tumor and tumor derived MV relate with cancer associated VTE and transplant human neutrophils to investigate the role of NETs in the VTE development in the next step.

並列關鍵字

cancer associated thromboembolism ； tissue factor ； extrinsic pathway ； intrinsic pathway ； tumor derived microvesicle

參考文獻

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