結合以氧化應激誘發癌細胞表現損傷相關分子與過濾擠壓法來生產癌細胞衍生奈米囊泡以製作治療性癌症疫苗

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鐘晉毅(Chin-Yi Chung) 查詢紙本館藏

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物理學系

論文名稱

結合以氧化應激誘發癌細胞表現損傷相關分子與過濾擠壓法來生產癌細胞衍生奈米囊泡以製作治療性癌症疫苗
(Therapeutic cancer vaccine made of cancer cell-derived nanovesicles produced by oxidative stress-induced expression of damage-associated molecular pattern and filter extrusion)

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摘要(中)

基於使用光動力療法（PDT）製備的癌症疫苗在動物實驗模型中，已被證實是治療癌症更有效的方法相較於其他癌細胞所衍生的癌症疫苗。似乎是由於光動力療法處理後的癌細胞產生高表現的熱休克蛋白70(HSP70)或其他與氧化應激誘導的損傷相關分子模式(DAMPs)，藉此所衍生的細胞膜奈米囊泡和細胞碎片使其效果更佳。為了開發出更具有治療潛力的癌症疫苗，從此實驗做延伸，我們使用不同氧化應激處理的方法去製備癌症疫苗，包括光動力療法使用標靶細胞不同位置的光敏藥劑、外加入H2O2應激處理和使用低溫氣壓式電漿（CAP）流應激處理。此外，我們將氧化應激處理後的癌細胞，使用過濾擠壓法(Filter Extrusion)產生奈米囊泡來製備癌症疫苗。藉由檢測癌症疫苗激活巨噬細胞產生一氧化氮(NO)濃度的測試、癌症疫苗激活的巨噬細胞與癌細胞共培養的毒殺測試。發現使用過濾擠壓法所產生的奈米囊泡通常比細胞自發性釋放的奈米囊泡更有效果。此外，與其他氧化應激源相比，使用低溫氣壓式電漿流處理所產生的疫苗是最有效的。結合低溫氣壓式電漿流處理以及過濾擠壓法所製備的癌症疫苗最具有治療癌症的潛力。

摘要(英)

Photodynamic therapy (PDT)-based cancer vaccine has been shown to be a more effective modality for treating cancer in animal models compared to other methods used to generate therapeutic cancer cell-derived vaccines. The higher efficacy seems to result from the generation of cell membrane nanovesicles or fragments that carry both cancer cell-specific antigens and a high surface content of HSP70 or other damage-associated molecular pattern molecules induced by oxidative stress. Aiming to develop more effective cancer vaccine along this direction, we explored cancer vaccines generated using different sources of oxidative stress, including photosensitizers that target different parts of the cells, externally added H2O2, and cold atmospheric plasma (CAP) jet. Furthermore, we explored cancer vaccines generated by using filter extrusion to produce nanovesicles from cancer cells after oxidative stress treatment. Through the tests of activating macrophages to secret NO, killing of cancer cells by co-cultured macrophages primed by the vaccines, and animal test, it is found that the vaccine based on nanovesicles generated by filter extrusion was generally more effective than that by spontaneous release of nanovesicles. In addition, the vaccine generated by using CAP jet treatment was the most effective compared to other sources of oxidative stress. The combination of CAP jet treatment and filter extrusion resulted in a vaccine that could lead to a total regression of the tumor in the mouse model, promising for human test.

關鍵字(中)

★ 奈米囊泡
★ 損傷相關分子模式
★ 低溫氣壓式電漿
★ 光動力療法
★ 過濾擠壓法

關鍵字(英)

★ Nanovesicle
★ Damage-associated molecular patterns
★ Cold atmospheric plasma jet
★ Photodynamic therapy
★ Filter extrusion

論文目次

中文摘要 i
Abstract ii
誌謝 iv
目錄 v
圖目錄 vi
第 1 章研究背景 1
1-1 癌症免疫療法簡介 1
1-2 光動力療法(PDT)疫苗介紹 3
1-3 熱休克蛋白70(HSP70)與細胞外囊泡(EVs) 6
1-4 PDT的光氧化應激與損傷相關分子模式(DAMPs) 7
1-5 低溫大氣式電漿流(CAP jet)介紹…… 8
1-6 過濾擠壓技術 9
1-7 巨噬細胞介紹 10
1-8 研究動機 11
第 2 章實驗材料與方法 13
2-1 實驗材料 13
2-2 氧化應激處理方法 14
2-3 癌細胞表徵熱休克蛋白70測量 17
2-4 自發性與過濾擠壓法產生奈米囊泡 17
2-5 使用NTA檢測奈米囊泡大小分布及數量 19
2-6 檢測疫苗激活巨噬細胞產生一氧化氮濃度方法 19
2-7 疫苗激活巨噬細胞與癌細胞共培養毒殺測量 20
2-8 動物試驗模型 20
第 3 章實驗結果與討論 22
3-1 自發性與過濾擠壓法奈米囊泡大小分佈與數量 22
3-2 不同光敏藥劑嵌合的癌細胞胞器 23
3-3 不同氧化應激處理的細胞影像 25
3-4 低溫大氣電漿流(CAP jet)與光動力療法(PDT)的熱影像 27
3-5 不同氧化應激方法產生熱休克蛋白70 (HSP70) 28
3-6 疫苗經過UV燈照射後的滅菌效果 29
3-7 各類疫苗激活巨噬細胞產生一氧化氮(NO)濃度比較 31
3-8 細胞染色測試各類疫苗激活巨噬細胞毒殺癌細胞比較 37
第 4 章結論與未來展望 40
第 5 章參考文獻 42

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指導教授

陳賜原

審核日期

2019-1-30

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