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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林淑華(Shu-Wha Lin) | |
dc.contributor.author | Chao-I Lin | en |
dc.contributor.author | 林昭怡 | zh_TW |
dc.date.accessioned | 2021-06-15T14:03:28Z | - |
dc.date.available | 2020-08-26 | |
dc.date.copyright | 2020-08-26 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-07 | |
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J Gastrointest Oncol, 2015. 6(3): p. E48-51. 53. Beauchemin, N., CEA Gene Family, in Encyclopedia of Cancer, M. Schwab, Editor. 2017, Springer Berlin Heidelberg: Berlin, Heidelberg. p. 870-874. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52020 | - |
dc.description.abstract | 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。 | zh_TW |
dc.description.abstract | 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. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T14:03:28Z (GMT). No. of bitstreams: 1 U0001-0708202016233100.pdf: 4340430 bytes, checksum: 003d5736ec7a3002b049720886481b3f (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 口試委員會審定書……………………………………………………………......I 誌謝………………………………………………………………………………..II 中文摘要…………………………………………………………………………..III 英文摘要…………………………………………………………………………..IV 總目錄……………………………………………………………………………..VI 圖目錄……………………………………………………………………………..X 表目錄……………………………………………………………………………..XI 第一章 緒論 1.1 止血作用(hemostasis)……………………………………………………...1 1.2 凝血機制及血纖維形成 (Blood coagulation and fibrin formation)………2 1.3 靜脈血栓栓塞…………………..…………………………………………..3 1.4癌症相關栓塞臨床意義及機制探討……………………………………….4 1.4.1 癌症相關栓塞臨床意義 1.4.2 癌症相關栓塞機制探討 1.5 誘發栓塞的動物模型………………………………………………………6 1.5.1 下腔靜脈阻斷(IVC stasis model)及狹窄(IVC stenosis model)模型 1.5.2 自由基誘發栓塞模型 1.5.3 雷射造成血管損傷誘發栓塞之模型 1.6 研究動機與目的…………………………………………………………....8 第二章 實驗材料與方法…………………………………………………………....9 2.1細胞培養與篩選穩定細胞株………….…………………………………….9 2.1.1細胞培養 2.1.2 建構AsPC-1組織因子剔除株 2.1.3篩選穩定細胞株 2.2 AsPC-1細胞野生株及組織因子剔除株增殖能力與移行能力……………10 2.2.1 細胞存活率試驗(MTT assay) 2.2.2 傷口癒合試驗(wound healing assay) 2.3 腫瘤微粒分離、定性及定量……………………………………………………………………..11 2.3.1腫瘤微粒分離 2.3.2腫瘤微粒的大小分布與濃度 2.3.3腫瘤微粒的型態 2.3.4 腫瘤微粒組織因子活性測定 2.4實驗動物……………………………………………………………………..12 2.5靜脈栓塞小鼠模型…………………………………………………………..13 2.5.1下腔靜脈狹窄手術 2.5.2 下腔靜脈採血 2.6 胰臟癌原位注射模型……………………………………………………….13 2.6.1腫瘤原位注射 2.6.2 心臟採血 2.7 免疫組織染色…………………………………………………………….....14 2.8 免疫螢光染色……………………………………………………………….15 2.9流式細胞儀…………………………………………………………………..15 2.9.1 細胞 2.9.2腫瘤微粒 2.10腫瘤微粒之螢光標定………………………………………………………16 2.11 數據分析與統計……………………………………………………….......16 第三章 實驗結果……………….………………………………...…………………17 3.1建立AsPC-1組織因子剔除(tissue factor, TF)株及篩選穩定表現細胞株...17 3.1.1建立AsPC-1組織因子剔除株 3.1.2篩選穩定表現細胞株 3.2 AsPC-1細胞野生株(TF+)及其組織因子剔除株(TF-)增殖及移行能力…17 3.3 AsPC-1細胞野生株與組織因子剔除株釋放之腫瘤微粒(microvesicle)定 性與定量……………………………………………………………………18 3.3.1 腫瘤微粒之大小分布與濃度 3.3.2腫瘤微粒之型態 3.3.3確認腫瘤微粒表現之膜蛋白 3.3.4 確認腫瘤微粒是否具備有活性之組織因子 3.4組織因子陽性腫瘤微粒對不同凝血路徑因子缺乏的小鼠造成之影響…19 3.4.1組織因子陽性腫瘤微粒對不同內在凝血路徑因子缺乏的小鼠產生血 栓重量的影響 3.4.2 小鼠注射組織因子陽性腫瘤微粒後休克死亡之病理原因探討 3.5 AsPC-1細胞野生株(TF+)及組織因子剔除株(TF-)注射NSG(Nod/Scid- Il2γ−/−)及A型血友病NSG小鼠(hemophilia A NSG, HA NSG)對腫瘤生 長、轉移及釋放腫瘤微粒的能力之影響…………………………………..20 3.5.1 腫瘤生長 3.5.2 腫瘤轉移 3.5.3 產生腫瘤的小鼠血漿中不含人類腫瘤微粒 第四章 討論…………………………………………………………………………23 4.1 AsPC-1野生株及組織因子剔除株腫瘤微粒注射到缺乏不同凝血因子小 鼠內產生血栓的結果差異…………………………………………………23 4.2腫瘤生長……………………………………………………………………24 4.3腫瘤轉移……………………………………………………………………24 4.4在胰臟原位注射模型中無法偵測到人類腫瘤微粒……………………....25 第五章 結論與展望…………………………………………………………………27 參考文獻……………………………….…………………………………………….28 圖………………………...…………...………………………………………………32 附表……………………………………………………………………………...…...52 附錄………………………………...……………………………...…………………53 | |
dc.language.iso | zh-TW | |
dc.title | 以小鼠模型探討腫瘤微粒在癌症相關靜脈血栓之角色 | zh_TW |
dc.title | Investigating the role of microvesicles in cancer-associated venous thrombosis by using mouse models
| en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 胡忠怡(Chung-Yi Hu),曾慶平(Ching-Ping Tseng),林淑容(Shu-Rung Lin) | |
dc.subject.keyword | 癌症靜脈栓塞,組織因子,內在途徑,外在途徑,腫瘤微粒, | zh_TW |
dc.subject.keyword | cancer associated thromboembolism,tissue factor,extrinsic pathway,intrinsic pathway,tumor derived microvesicle, | en |
dc.relation.page | 54 | |
dc.identifier.doi | 10.6342/NTU202002651 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-08-10 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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