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研究生:邊士純
研究生(外文):Shih-Cheung Bian
論文名稱:循環腫瘤細胞檢測針的生物相容性調查
論文名稱(外文):The investigation of biocompatibility of circulating tumor cell needle detector
指導教授:翁文慧
口試委員:吳慧中潘同明馮思中
口試日期:2016-07-24
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:化學工程與生物科技系生化與生醫工程碩士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
畢業學年度:104
中文關鍵詞:血液相容性測試毒性測試生物相容性生物感測器
外文關鍵詞:Hemocompatibility testToxicity testBiocompatibilityBiosensor
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利用表面修飾技術所製之循環腫瘤細胞 (Circulating tumor cells, CTCs) 檢測針,為一種活體內的CTCs的篩檢平台,而本篇針對本實驗室所研製之CTCs檢測針進行生物相容性 (Biocompatibility) 檢測,評估應用於活體中之生物相容性。本研究根據國際標準組織 (International Organization for Standardization,ISO) 標準,針對CTCs檢測針的特性,建立一套快速、有效並符合測試條件的生物相容性檢驗方法,包括細胞毒性測試、血液相容性測試及動物實驗測試之三大架構。體外細胞毒性測試中,以CTCs檢測針萃取液對L929小鼠纖維母細胞進行共培養並觀察其形態學(Morphology) 及MTS細胞存活率的影響,並藉由RAW264.7小鼠巨噬細胞株進行發炎反應測試;血液相容性測試為將CTCs檢測針浸泡於ICR小鼠血液中並檢測凝血酶原時間 (Prothrombin time, PT) 和部分血酶原時間 (Activated partial thromboplastin time, APTT),確認對凝血的影響;動物實驗採用ICR小鼠進行內皮刺激性測試和系統急性毒性測試進行評估。藉由此一系列之檢測方法,證實循環腫瘤細胞檢測針具有良好的生物相容性,以期未來能儘早應用於人體癌症的篩檢中。本篇研究建立了一套應用於活體內之醫療器材的檢驗方法,可供未來研發之不同的生物檢測器的評估方法參考。
The needle type detector which the circulating tumor cells (CTCs) employ the technique of surface modification is a capturing method of CTCs in a living body. In this study, a close inspection is carried on to evaluate the biocompatibility of the developed needle type dector. The experiment is developed under the process which conforms the regulation of the International Organization for Standardization (ISO), including the cytotoxicity test, hemocompatibility test and in vivo test. First, with the needle type detector of CTCs, cytotoxicity test examines the effect that the extract solution places upon the L929 fibroblast cells, viability test by MTS assay and inspect whether RAW264.7 macrophage cells react in inflammatory response. Secondly, in the part of hemocompatibility test, the needle type detector of CTCs is soaked into the blood of the ICR mice, and both Prothrombin time (PT) and Activated partial thromboplastin time (APTT) were observed. Finally, animal skin irritation test of ICR mice observes whether or not the extract solution has a direct effect on the skin tissue. In addition, the test of acute systemic toxicity observes the effect after penetrating the type needle detector into the celiac part of the ICR mice. By means of the series of tests, the needle type detector of CTCs has an excellent biocompatibility is proven and that with expectancy, we wish to see the application of the detector on the screening of human body soon. The study develops an immaculate and suitable inspecting method which provides future experiment a model.
摘 要 I
英文摘要 III
誌 謝 V
表目錄 XI
圖目錄 XII
第一章 導論 1
1.1 生物相容性 1
1.1.1醫療器材之生物相容性測試的重要性 1
1.1.2醫用器材測試項目之選擇與評估 2
1.2 醫療器材之生物相容性測試相關法規 5
1.2.1 ISO與醫療器材之關係 5
1.2.2 ISO10993醫療器材之生物相容性試驗法規 5
1.3 循環腫瘤細胞檢測針 8
1.3.1 循環腫瘤細胞 8
1.3.2 循環腫瘤細胞檢測針的優勢 9
1.3.3 循環腫瘤細胞檢測針 9
第二章 研究目標 10
2.1 循環腫瘤細胞檢測針之生物相容性評估 10
2.2循環腫瘤細胞檢測針生物相容性測試項目的建立 11
第三章 材料與方法 14
3.1 材料 14
3.1.1 L929小鼠成纖維細胞培養 14
3.1.2 RAW264.7小鼠巨噬細胞培養 14
3.2實驗方法 15
3.2.1培養基的配置 15
3.2.2細胞解凍 15
3.2.3細胞繼代 15
3.2.4細胞凍存 16
3.2.5細胞計數 16
3.2.6測試材料萃取液製備 17
3.2.6.1實驗原理 17
3.2.6.2實驗條件 18
3.2.6.3實驗步驟 18
3.2.7細胞型態學觀察 19
3.2.7.1實驗原理 19
3.2.7.2實驗條件 19
3.2.7.3實驗步驟 19
3.2.8細胞存活率測試 20
3.2.8.1實驗原理 20
3.2.8.2實驗條件 21
3.2.8.3實驗步驟 21
3.2.9細胞發炎性測試 21
3.2.9.1實驗原理 21
3.2.9.2 NO產生量測試 23
3.2.9.2.1實驗原理 23
3.2.9.2.2實驗條件 25
3.2.9.2.3實驗步驟 25
3.2.9.3發炎反應之ROS產生量測試 25
3.2.9.3.1實驗原理 25
3.2.9.3.2實驗條件 26
3.2.9.3.3實驗步驟 26
3.2.9.4發炎反應之NF-kB產生量測試 26
3.2.9.4.1實驗原理 26
3.2.9.4.2實驗條件 27
3.2.9.4.3實驗步驟 27
3.2.10內皮刺激性測試 27
3.2.10.1實驗原理 27
3.2.10.2實驗條件 28
3.2.10.3實驗步驟 28
3.2.11組織石蠟包埋和切片 29
3.2.11.1組織石蠟包埋和切片原理 29
3.2.11.2實驗條件 31
3.2.11.3實驗步驟 31
3.2.12蘇木素-伊紅 (H&E) 組織染色 32
3.2.12.1實驗原理 32
3.2.12.2實驗條件 32
3.2.12.3實驗步驟 32
3.2.13系統注射試驗 33
3.2.13.1實驗原理 33
3.2.13.2實驗條件 33
3.2.13.3實驗步驟 34
3.2.14血液復鈣測試 35
3.2.14.1凝血酶原 (Prothrombin time,PT) 時間測試 35
3.2.14.1.1實驗原理 35
3.2.14.1.2實驗條件 36
3.2.14.1.3實驗步驟 36
3.2.14.2部分凝血活酶試驗 (APTT) 37
3.2.14.2.1實驗原理 37
3.2.14.2.2實驗步驟 37
第四章 實驗結果 38
4.1循環腫瘤細胞檢測針萃取液對L929細胞型態生長影響結果 38
4.2循環腫瘤細胞檢測針萃取液對L929細胞存活率測試結果 41
4.3細胞毒性測試-發炎反應測試結果 43
4.3.1環腫瘤細胞檢測針對RAW264.7細胞發炎反應-NO濃度測試 43
4.3.2環腫瘤細胞檢測針對RAW264.7細胞發炎反應-ROS濃度測試 45
4.3.3循環腫瘤細胞檢測針萃取液對RAW-BLUETM細胞發炎反應-NF
-KB濃度測試 45
4.4循環腫瘤細胞檢測針對ICR小鼠之內皮刺激性試驗結果 47
4.5循環腫瘤細胞檢測針對ICR小鼠之內皮組織H&E染色結果 50
4.6急性系統毒性測試小鼠行為觀察結果 51
4.7急性系統毒性測試小鼠肝臟組織生理學變化 52
4.8 血液復鈣測試結果 52
4.8.1 PT測試結果 52
4.8.2 APTT測試結果 52

第五章 結果討論 54
5.1循環腫瘤細胞檢測針之細胞毒性測試結果 54
5.2循環腫瘤細胞檢測針發炎反應測試部分結果討論. 55
5.3循環腫瘤細胞檢測針ICR小鼠內皮刺激性測試結果討論. 55
5.4循環腫瘤細胞檢測針ICR小鼠急性系統毒性測試 56
5.5循環腫瘤細胞檢測針之血液復鈣測試結果 56
參考文獻 57
附錄一 61
附錄二 62
附錄三 63
附錄四 64
附錄五 65
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