癌細胞是一種不正常增生的突變細胞，不受控制迅速增殖的腫瘤團塊限制了周邊血液以及氧氣供應，使其局部氧氣含量明顯低於健康組織，出現了｢缺氧｣的狀態，幾乎所有實體腫瘤都具有相似的微環境特徵。近年來的研究顯示，腫瘤缺氧的狀態下會刺激缺氧誘導因子HIF(hypoxia-inducible factor)表現，並造成許多影響，包括:血管生成、腫瘤轉移擴散、多重抗藥性產生、調節細胞增殖及抑制細胞凋亡。進而使腫瘤細胞延長存活並產生抗藥性及影響治療成功率。
本研究針對不同病人、腫瘤缺氧狀況彼此的差異，建立一項仿缺氧微環境之微型晶片，利用聚二甲基矽氧烷PDMS(Polydimethylsiloxane) 對於氣體的通透性，使用還原劑亞硫酸鈉還原吸收細胞培養區的的氧分子，在化學還原劑不與細胞直接接觸的前提下同時形成四種不同的氧氣含量。使用生物相容水膠材料GelMA作為癌細胞A549的3D生長支架，比較不同缺氧環境下對於免疫細胞Jurkat的遷移能力影響以及使用化療藥物Pemetrexed合併免疫藥物Pembrolizumab在這些環境中的治療效果。
實驗結果顯示，氧分子含量最低之腔室[O2]Chamber1=2%的免疫細胞遷入數量最少，並依序遞增到正常培養箱【O2】=20%，接著免疫細胞遷入數量與免疫治療藥物效果成正比，推測是透過低氧環境抑制免疫細胞之活性，再進而降低藥物療效。

Cancer cells are abnormal, mutated cells that undergo uncontrolled and rapid proliferation, forming tumor masses that restrict the supply of blood and oxygen to the surrounding tissues. This leads to significantly lower oxygen levels compared to healthy tissues, resulting in a state of "Hypoxia," which is a common micro-environmental characteristic in nearly all solid tumors. Recent research has shown that the hypoxic conditions in tumors stimulate the expression of Hypoxia-Inducible Factor (HIF) and have various effects, including promoting angiogenesis, facilitating tumor metastasis, inducing multidrug resistance, regulating cell proliferation, and inhibiting apoptosis. This ultimately allows tumor cells to survive longer, develop drug resistance, and impact the success rates of chemotherapy and radiotherapy.
In this study, we aimed to establish a microchip mimicking the hypoxic tumor microenvironment to investigate the differences in hypoxia among different patients and tumor conditions. We designed a microchip using PDMS (polydimethylsiloxane), which allows gas permeability, and employed sodium bisulfite as a reducing agent to reduce oxygen molecules within the cell culture chambers. Four different oxygen levels were generated simultaneously without direct contact with the cells. We utilized a biocompatible hydrogel material called GelMA as a 3D growth scaffold for cancer cells(A549.
We compared the impact of different hypoxic environments on the migration capability of immune cells (Jurkat) and assessed the therapeutic efficacy of chemotherapy drug Pemetrexed in combination with the immune drug Pembrolizumab within these environments.
The experimental results revealed that the chamber with the lowest oxygen concentration [O2]Chamber1=2% had the fewest immune cells migrating into it. The number of immune cells gradually increased up to the normal culture condition [O2] = 20%. Furthermore, the number of immune cells migrating and the effectiveness of immunotherapy were positively correlated, suggesting that the low oxygen environment inhibited the activity of immune cells, subsequently reducing the efficacy of the drugs.

ABSTRACT I
摘要 II
致謝 III
目錄 V
圖表目錄 VII
第一章 緒論 1
1.1 前言 1
1.2 研究動機與目的 3
1.3 研究背景 4
1.3.1 生醫微機電與實驗室晶片 4
1.3.2 癌症 6
1.3.3 缺氧(Hypoxia) 8
1.4 文獻回顧 9
1.4.1 濃度梯度產生器與組合混合器 9
1.4.2 生物相容性水膠支架 11
1.4.3 氣體含量控制方法 12
1.4.4 缺氧環境與細胞關係 15
1.4.5 HIF蛋白的作用及機制 16
1.4.6 PD-1/PD-L1功能及路徑 18
1.4.7 化療合併免疫藥物治療 19
第二章 系統理論與晶片設計 21
2.1 系統理論 21
2.1.1 微流體晶片設計理論 21
2.1.2 微型混合理論 24
2.2 擴散理論 25
2.3 氧張力測量方法 26
2.3.1 克拉克式電極 27
2.3.2 發光光學感測器(Stern-Volmer kinetic relationships) 27
第三章 微流道晶片設計與製程 28
3.1 微流體晶片設計 28
3.1.1 詳細設計及功能介紹 28
3.2 製作流程 32
3.2.1 微流道晶片母模製程 32
3.2.2 微流道晶片製程 34
3.3 製程結果 36
第四章 實驗材料與方法 37
4.1 細胞培養 37
4.1.1 細胞繼代方法（A549, 貼附型）： 37
4.1.2 細胞繼代方法（Jurkat, 懸浮型）： 38
4.2 生物相容性水膠GELMA 38
4.3 IL-1 BETA 重組蛋白 40
4.4 化療藥物以及免疫療法藥物 40
4.5 化學還原劑 40
4.6 氧分子探針 41
4.7 細胞螢光染劑 42
4.8 細胞死活螢光染劑 42
4.9 細胞存活率分析(CCK-8 ASSAY) 43
4.10 實驗設備 45
4.11 晶片操作流程 46
第五章 實驗結果與討論 47
5.1 混合模組及晶片測試 47
5.2 腔室中含氧量分析 47
5.3 GELMA曝光時間存活率測試 48
5.4 癌細胞在不同氧含量環境之生存率分析 50
5.5 PEMETREXED 藥物對於A549半抑制濃度 51
5.6 免疫細胞之遷移能力及合併藥物治療效果 52
第六章 結論 55
第七章 參考文獻 57

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