腫瘤微環境在腫瘤的生長過程中扮演的極為重要的角色，在這個特別的環境中，存在著缺氧、低pH以及高壓的問題，並且由癌細胞特殊的代謝模式－Warburg Effectn所造成乳酸大量堆積，使得腫瘤微環境形成酸中毒現象，其酸鹼值呈現pH 6.7-7.2。並且其代謝模式的副產物－乳酸，更會進一步調節免疫細胞的功能並促進腫瘤的侵襲及轉移。因此，將腫瘤微環境中的乳酸堆積現象消除將成為本研究之重點。本研究將設計一具有生物相容性之甲基纖維素（Methylcellulose, MC）凝膠載體包覆乳酸氧化酶（Lactate Oxidase from Aerococcus viridians, LOX）開發成具有溫度敏感性的藥物載體系統，在腫瘤微環境中進行持續性的釋藥，以緩解腫瘤微環境中乳酸大量堆積的現象。根據各項研究結果得知，在三天的作用下，包覆LOX的水凝膠經過吸水膨脹後，將LOX釋放於環境中，可以在pH 6.7的環境下氧化91.7 %的乳酸。除此之外，輔助腫瘤生長的M2型態腫瘤相關巨噬細胞轉換至抑制腫瘤生長的M1型態巨噬細胞其轉換效率以及M1型態巨噬細胞特有的NO和成功能分別增加16.46 %及18.97 %，並且由M2型態轉換為M1型態之巨噬細胞經實驗證實具有擊殺腫瘤細胞之功能。本研究之各項實驗結果皆顯示本研究設計之甲基纖維素水凝膠包覆乳酸氧化酶經過長時間的作用下可以達到回復自體免疫系統擊殺癌細胞的功能。

Deregulated proliferation of tumor is generally associated with altered energy metabolism. A high rate of anaerobic glycolysis in solid tumors contributes to an acidification of pH to 6.7–7.2 in the tumor microenvironment due to lactate accumulation. Lactate modulates immune cell function and promotes tumor invasion and metastasis. However, a particular challenge is to clean lactate in tumor microenvironment. Here we report exploitation of tumor lactate production in designing a methylcellulose (MC) hydrogel incorporating lactate oxidase (LOX), and MC-LOX hydrogel was used controlled the degree of swelling, water uptake and subsequent degradation of the hydrogels and release rate of LOX. After 3 days, MC-LOX hydrogel was exemplified by achieving 91.7 % lactate consumption at pH 6.7, compared with control groups. Furthermore, the conversion efficiency and unique NO synthase function of tumor-supportive macrophages (M2) to tumor-suppressive macrophages (M1) was increased 16.46 % and 18.97 %, respectively. This is relevant to optimizing parameters for immune medicine applications.

論文審定書 i
誌謝 ii
中文摘要 iii
英文摘要 iv
目錄 v
圖目錄 viii
表目錄 x
第一章、前言 1
1.1 癌症 4
1.1.1 概況 5
1.1.2 治療方式 5
1.1.2.1 外科手術治療 5
1.1.2.2 化學治療 6
1.1.2.3 放射線治療 7
1.1.2.4 標靶治療 8
1.1.2.5 免疫治療 9
1.2 腫瘤微環境 11
1.2.1 缺氧環境（Hypoxia） 12
1.2.2 低pH環境（Low pH） 13
1.2.3 高壓環境（High Pressure） 13
1.2.4 腫瘤細胞對特殊環境之適應 14
1.3 腫瘤微環境中的免疫機制－腫瘤相關巨噬細胞（Tumor-associated Macrophages, TAM） 14
1.3.1 M1型態腫瘤相關巨噬細胞 16
1.3.2 M2型態腫瘤相關巨噬細胞 17
1.3.3 酸性環境對腫瘤相關巨噬細胞之影響 18
1.4 乳酸氧化酶（Lactate Oxidase, LOX） 19
1.5 甲基纖維素（Methyl Cellulose）溫敏性水凝膠 20
1.6 研究動機與目的 21
第二章 材料方法 23
2.1 儀器與藥品 23
2.2 甲基纖維素凝膠製備 25
2.2.1 0.5倍磷酸鹽緩衝溶液（Phosphate Buffered Saline）配製 25
2.2.2 16 %（W‧V-1）甲基纖維素粉末 26
2.3 L-乳酸調整培養液的酸鹼值 26
2.4 小鼠巨噬細胞RAW 264.7培養 27
2.4.1 腫瘤相關巨噬細胞M1型極化 27
2.4.2 腫瘤相關巨噬細胞M2型極化 28
2.5 細胞免疫螢光定量分析 28
2.5.1 細胞收取 28
2.5.2 螢光抗體染色 28
2.6 細胞免疫螢光定性分析 29
2.6.1 細胞樣品準備 29
2.6.2 螢光抗體染色 29
2.7 MTS細胞活性測試（MTS assay） 30
2.8 LIVE/DEAD細胞活性測試（LIVE/DEAD Viability/Cytotoxicity Kit） 31
2.9 LOX氧化乳酸能力檢測 32
2.10 腫瘤相關巨噬細胞M1型功能測試 32
2.11 體外實驗測試腫瘤相關巨噬細胞對腫瘤細胞之影響 34
第三章 結果與討論 35
3.1 實驗流程圖 35
3.2 L-乳酸調整培養液之酸鹼值滴定 36
3.3 材料的生物相容性測試 36
3.4 包覆LOX之水凝膠其藥物釋放能力檢測 38
3.5 酸性環境對於小鼠巨噬細胞RAW 264.7之細胞活性測試 39
3.6 小鼠巨噬細胞RAW 264.7極化 40
3.7 酸性環境對於M1及M2型小鼠巨噬細胞RAW 264.7極化之影響 42
3.8 以乳酸調控培養液酸鹼值對於M0型小鼠巨噬細胞RAW 264.7之影響 44
3.9 不同酸鹼值之環境於長時間下對M1型小鼠巨噬細胞RAW 264.7之影響 46
3.10 不同酸鹼值之環境於長時間下對M2型小鼠巨噬細胞RAW 264.7之影響 47
3.11 LOX水凝膠改變培養液乳酸含量後對小鼠巨噬細胞RAW 264.7之影響 49
3.12 M1型小鼠巨噬細胞RAW 264.7功能測試 51
3.13 體外實驗測試腫瘤相關巨噬細胞對腫瘤細胞之影響 54
第四章 結論與討論 55
參考文獻 57

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