惡性腫瘤 (Malignant Tumor) 即癌症連續三十六年成為台灣人致死因子之首，然而癌症的轉移是癌症造成死亡的最主要因素。惡性腫瘤透過循環腫瘤細胞 (Circulating Tumor Cells, CTC) 從原位癌化器官組織脫離後穿過血管壁進入血液或淋巴循環，當循環腫瘤細胞抵達將發生癌症轉移器官時，會穿透微血管壁，進入該器官的組織，然後增生出更多的腫瘤細胞而形成新腫瘤，這種遠端轉移是導致腫瘤患者死亡的主要原因。
轉移前微環境(Pre-Metastatic Niche，PMN) 為一種有利於原發腫瘤轉移的次級器官環境，透過建立一個仿生組織向的轉移前微環境可以有效引誘癌症細進行移轉即增生行為，透過微環境的建立以及細胞實驗可以有效觀察癌化細胞轉移的趨勢以研擬抗轉移的治療策略。本研究致力於建立一基於水膠材料之新穎仿生微環境模型晶片，以探討物理性因素對正常組織中形成轉移前微環境的影響。本研究主要分兩種微環境平台，三維海藻酸納雙層中空水膠管(Alginate Hydrogel Tube, AHT)；Transwell 支架水膠薄片。透過改變兩種平台中水膠材料的物理參數如硬度、表面粗糙、孔隙結構等以此模擬不同組織引誘因子，其中AHT模型更可以改變中空管內壓力、流速、通入物質以模擬血管，再以ATP測定法測量定量細胞向性以觀察細胞遷移狀況。兩個平臺在乳腺癌細胞株遷移實驗中皆發現高硬度且粗糙度的水膠材料對於乳癌細胞株有著更好的吸引效果。未來期待藉由本研究提出之此微環境模型晶片探討出臨床上癌症細胞轉移的機制並對試藥及治療上有顯著貢獻。

Malignant tumor, so called cancer have been the first death cause in Taiwan for 36 years. However, the cancer cell metastasis is the main factor that causing death. The cancer cell will enter the blood circulation system or lymphatic system from the carcinoma in situ by penetrating into vascular wall via circulating tumor cells (CTC).
CTCs will penetrate microvascular wall to the metastasis tissue or organ and grow as a new tumor. This kinds of cancer cell migration is the leading cause of death in cancer patients.
Pre-metastatic Niche (PMN) is a secondary organ environment that is conducive to primary tumor metastasis. Through the construction of a pre-metastatic environment could induce the cancer cell metastasize and growth. We could develop a effectively treatment strategies by observing the trend of the cancer cell metastasizing with PMN.
In this research, we aim to establish a novel biomimetic microenvironment chip to explore the influence of biophysical factor to the cancer cell metastasize. This research provides two kinds of PMN platform, three-dimensional Alginate Hydrogel Tube (AHT); Transwell-based Alginate Hydrogel (TAH). We would observe the cell metastasis trend by changing the physical factor in hydrogel such as stiffness, surface roughness, pore structure. Further more, we could control the pressure, flow rate and with inducing material in AHT model. Quantitative cell tropism was measured by ATP assay to observe cell migration status. Both platforms have found that high-hardness and roughness of the hydrogel material has a better attraction effect on breast cancer cell lines in the migration experiment. In the future, it is expected that the microenvironment model proposed in this study will explore the mechanism of clinical cancer cell metastasis and make significant contributions to the drug and treatment.

目錄
摘要 I
Abstract II
致謝 III
目錄 IV
表目錄 VII
圖目錄 VIII
第一章 緒論 1
1.1 研究背景前言 1
1.2研究動機 3
第二章 文獻回顧 5
2.1 水膠 5
2.3水膠表面 8
2.4 三維管狀水膠 12
2.5 腫瘤轉移前利基與腫瘤微環境 16
2.6腫瘤轉移模型 17
2.6.1模擬乳腺癌轉移至骨轉移細胞-細胞反應之微流體3D體外模型 17
2.6.2模擬乳腺癌轉移至骨轉移流體壓力之微流體3D體外模型 18
第三章 實驗設計與規劃 20
3.1 實驗平臺設計 20
3.1.1 雙層中空海藻酸鈉水膠微管(Alginate Hydrogel Tube AHT)遷移模型 20
3.1.2 基於Transwell之海藻酸鈉水膠遷移模型 21
3.1.3 循環式海藻酸鈉水膠微管微環境系統 21
3.2海藻酸鈉水膠製程 23
3.2.1前置藥品製備 23
3.2.2 三維中空海藻酸鈉水膠微管 24
3.2.3 基於Transwell之海藻酸鈉水膠薄片 25
第四章 實驗操作與流程 26
4.1海藻酸鈉水膠處理過程 26
4.1.1水膠滅菌處理 26
4.1.2水膠脫水處理 26
4.2細胞培養微環境操作 26
4.2.1細胞培養技術 26
4.2.2 細胞培養液準備 27
4.2.3細胞培養 27
4.2.4細胞螢光染色 28
4.2.5細胞活力測定 28
4.3實驗儀器與藥品 29
4.3.1細胞培養實驗 29
4.3.2 海藻酸鈉水膠微管(AHT)實驗 30
4.3.3基於Transwell之TAH實驗 31
4.3.4循環式微環境系統實驗 32
第五章 實驗結果與討論 34
5.1 三維海藻酸鈉水膠微管 34
5.1.1 三維海藻酸鈉水膠微管製程 34
5.1.2 水膠管通細胞遷移測試 35
5.2 AHT模型細胞遷移測試 36
5.2.1 AHT細胞遷移模型架設 36
5.2.2 水膠表面硬度之物理因素改變 37
5.2.2表面硬度之改變對於細胞遷移之影響性 40
5.2.3水膠表面形貌之物理因素改變 41
5.2.4表面形貌之改變對於細胞遷移之影響性 45
5.2.5綜合物理因素改變對於細胞遷移之影響性 47
5.3 TAH模型細胞遷移測試 48
5.3.1 TAH細胞遷移模型架設 48
5.3.2 TAH模型細胞遷移測試 49
5.3.3 TAH模型藥物測試 49
5.3.4 3D-Printing海藻酸鈉水膠片應用於TAH模型 52
5.4 AHT與TAH細胞遷移模型比較 56
5.5 循環式微環境系統測試模擬 56
第六章 結論 58
6.1微環境之機械強度因素 58
6.2水膠表面形貌因素 58
第七章 未來工作 59
參考文獻 61

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