微生物機器人：利用金屬有機骨架材料封裝大腸桿菌誘導癌症細胞焦亡

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孫煜程(Yu-Cheng Sun) 查詢紙本館藏

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化學學系

論文名稱

微生物機器人：利用金屬有機骨架材料封裝大腸桿菌誘導癌症細胞焦亡
(Microbial Robots: Inducing Cancer Cell Pyroptosis with Encapsulated E. coli in Metal-Organic Framework)

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至系統瀏覽論文 (2029-8-1以後開放)

摘要(中)

癌症一直是全球公共衛生的重大挑戰，傳統治療方法如手術、化療和放療往往會引發強烈副作用。而細菌介導(Bacteria-mediated)的癌症治療作為一種有前景的替代方案逐漸嶄露頭角，但仍需克服在常規滅菌下維持目標細菌的活性，以及避免對人體有害的內毒素釋放的問題。因此，研究人員正探索新的藥物載體，以克服上述問題並提升療效。金屬有機骨架材料（MOFs）因為其多樣性及穩定性而備受關注。本研究展示了將大腸桿菌封裝在一種MOFs類型—類沸石咪唑骨架材料-90（ZIF-90）的過程，藉由封裝可以使細菌UV滅菌下保有活性，且可以規避人體內抗生素的侵襲，除此之外也可以防止細菌直接暴露於血液循環中引發強烈免疫反應。體外實驗表明，E. coli@ZIF-90使乳腺癌細胞僅剩47%的存活率，原因是細菌外膜上的脂多醣（LPS）誘導的發炎反應。本研究透過西方墨點法驗證E. coli@ZIF-90能誘導乳腺癌細胞焦亡（Pyroptosis），且在E. coli@ZIF-90的處理下細胞焦亡路徑的各蛋白質表現量與對照組均有顯著差異；相反地在正常細胞中沒有細胞焦亡的發生。並透過感應耦合電漿質譜檢測在E. coli@ZIF-90處理後乳腺癌細胞內鋅離子的濃度高達1.15 mg/L，而正常細胞僅有0.43 mg/L，兩者之間有顯著差異（P < 0.05）。最後利用顯微鏡觀察到在E. coli@ZIF-90處理後乳腺癌細胞內部大腸桿菌的數量和正常細胞有顯著差異（P < 0.0001），上述體外實驗證明癌症細胞比起正常細胞更能夠吞噬E. coli@ZIF-90，這突顯了治療的專一性。動物實驗進一步驗證在E. coli@ZIF-90處理之下，抑制了72%癌症細胞之結節面積。這些結果展現了ZIF-90作為細菌介導癌症治療載體的潛力。在酸性腫瘤環境中，E. coli@ZIF-90選擇性釋放出細菌，在缺氧環境下細菌將定植於腫瘤中。這不僅能誘導癌細胞的焦亡，還能活化免疫系統攻擊腫瘤。這項研究在癌症治療研究中做出了重要貢獻，提供了不一樣的治療可能性。
總之，將大腸桿菌封裝在ZIF-90中是一種新穎且有效的標靶細菌介導癌症治療策略，在體外和體內實驗均顯示抑制癌症腫瘤的結果。

摘要(英)

Cancer remains a significant global health challenge, with traditional treatments like surgery, chemotherapy, and radiation often causing severe side effects. Bacterial-mediated cancer therapy is emerging as a promising alternative, though it faces challenges such as maintaining bacterial viability during sterilization and avoiding harmful endotoxin release. Researchers are exploring new drug delivery materials to enhance the efficacy of bacterial therapies, and Metal-Organic Frameworks (MOFs) have gained attention for their versatility and stability. This study demonstrates the encapsulation of E. coli within Zeolitic Imidazolate Framework-90 (ZIF-90), a type of MOFs. Encapsulating bacteria within ZIF-90 allows them to retain activity under UV sterilization and evade antibiotic attacks within the human body. Furthermore, this method prevents the bacteria directly exposed to the bloodstream, thereby avoiding a strong immune response .In vitro experiments showed that E. coli@ZIF-90 reduces the survival rate of breast cancer cells to 47%, attributed to lipopolysaccharides (LPS) on the bacterial membrane inducing inflammatory responses. Simultaneously, Western blot analysis verified that E. coli@ZIF-90 can induce pyroptosis in breast cancer cells. The expression levels of proteins involved in the pyroptosis pathway were significantly different in breast cancer cells treated with E. coli@ZIF-90 compared to the control group. In contrast, pyroptosis hasn’t been occured in normal cells. Using inductively coupled plasma mass spectrometry (ICP-MS), it was detected that the concentration of zinc ions in breast cancer cells after E. coli@ZIF-90 treatment reached up to 1.15 mg/L, compared to 0.43 mg/L in normal cells, showing a significant difference (P < 0.05). Microscopic observations revealed a significant difference (P < 0.0001) in the amount of internalized E. coli between breast cancer cells and normal cells after E. coli@ZIF-90 treatment. In vivo experiments confirmed that treatment with E. coli@ZIF-90 inhibited 72% of the cancer cell nodule area, underscoring the potential of ZIF-90 as a carrier in bacterial-mediated cancer therapy. In the acidic tumor environment, E. coli@ZIF-90 selectively releases bacteria that colonize the tumor in a hypoxic environment, inducing pyroptosis in cancer cells and activating the immune system to attack the tumor. This study offers innovative approaches that could lead to breakthroughs in cancer therapy.
In summary, encapsulating E. coli within ZIF-90 presents a novel and effective strategy for targeted bacterial-mediated cancer therapy, showing promising results both in vitro and in vivo.

關鍵字(中)

★ 金屬有機骨架材料
★ 微生物封裝
★ 細菌癌症療法
★ 乳腺癌細胞
★ 細胞焦亡
★ 脂多醣

關鍵字(英)

★ Metal-organic Frameworks
★ Microbe Encapsulation
★ Bacteria-mediated Cancer Therapy
★ Breast Cancer Cells
★ Pyroptosis
★ Lipopolysaccharide

論文目次

中文摘要 i
Abstract iii
致謝 v
目錄 vi
圖目錄 xi
表目錄 xvi
1 第一章緒論 1
1-1 金屬有機骨架材料 1
1-1-1金屬有機骨架材料 1
1-1-2類沸石咪唑骨架材料 2
1-1-3類沸石咪唑骨架材料-90 3
1-2 癌症 6
1-2-2乳腺癌簡述 7
1-2-3常見的癌症治療方法 7
1-3 細菌癌症療法 9
1-3-1細菌癌症療法簡述 9
1-3-2細菌癌症療法困境 12
1-3-3大腸桿菌(Escherichia coli, E. coli) 12
1-3-4脂多醣(Lipopolysaccharide, LPS) 15
1-4 癌症細胞株與正常細胞株 17
1-4-1鼻咽癌細胞（NPC-TW01） 17
1-4-2胃腺癌細胞株（AGS） 17
1-4-3小鼠乳腺癌細胞（4T1） 18
1-4-4人類乳腺癌細胞（Hs 578T） 18
1-4-5人類正常乳腺上皮細胞（H184） 18
1-5 細胞死亡 20
1-5-1細胞凋亡（Apoptosis） 20
1-5-2細胞焦亡（Pyroptosis） 20
1-6 研究動機與目的 22
2 第二章實驗部分 24
2-1 實驗藥品與設備 24
2-2 實驗儀器原理 30
2-2-1粉末Ｘ射線繞射儀 (Powder X-ray Diffractometer, PXRD) 30
2-2-2螢光顯微鏡（Fluorescence Microscope） 31
2-2-3共軛焦顯微鏡（Confocal Microscopy） 33
2-2-4紫外/可見光光譜儀 34
2-2-5西方墨點法（Western Blotting） 34
2-2-6酶聯免疫吸附測定（Enzyme-linked Immunosorbent Assay, ELISA） 35
2-2-7細胞存活率測試（Cell Viability Assay） 36
2-2-8感應耦合電漿質譜儀（Inductively Coupled Plasma Mass Spectrometry, ICP-MS） 37
2-3 實驗步驟 38
2-3-1合成類沸石咪唑骨架材料-90（ZIF-90） 41
2-3-2大腸桿菌之培養步驟 41
2-3-3合成類沸石咪唑骨架材料-90封裝大腸桿菌及脂多醣之生物複合材料（E. coli@ZIF-90, ClearColi@ZIF-90, LPS@ZIF-90） 42
2-3-4 類沸石咪唑骨架材料-8塗覆大腸桿菌(E .coli⸦ZIF-8)之合成步驟 44
2-3-5細胞培養 44
2-3-6動物實驗 44
2-3-7組織分析 45
2-3-8西方墨點法（Western Blotting） 46
2-3-9酶聯免疫吸附測定（Enzyme-linked Immunosorbent Assay, ELISA） 51
2-3-10 細胞存活率實驗 52
2-3-11細胞吞噬染色實驗 52
2-3-12 溶酶體螢光探針（Lysotracker） 53
2-4 統計分析 54
3 第三章結果與討論 55
3-1 ZIF-90與E. coli@ZIF-90之相關鑑定 55
3-1-1粉末X射線繞射儀之鑑定結果 55
3-1-2共軛焦顯微鏡之成像結果 56
3-2 E. coli@ZIF-90抵抗滅菌程序及抗生素 58
3-2-1 E. coli@ZIF-90抗UV實驗 58
3-2-2 E. coli@ZIF-90抵擋抗生素之實驗 59
3-3 巨噬細胞對於E. coli@ZIF-90的發炎反應 61
3-4 生物複合材料對於乳腺癌細胞株之存活率測試 63
3-5 生物複合材料對於乳腺癌細胞之發炎反應測試 65
3-6 ClearColi@ZIF-90與LPS@ZIF-90之相關鑑定 67
3-7 生物複合材料對於乳腺癌細胞株之存活率測試 69
3-8 生物複合材料對於正常乳腺細胞之細胞存活率測試 72
3-9 細胞對於生物複合材料之吞噬能力測試 74
3-9-1細胞內鋅離子濃度之測量 74
3-9-2在顯微鏡下觀測細胞吞噬效果 75
3-10 脂多醣誘導細胞焦亡反應之驗證 80
3-11 半胱天冬酶抑制劑對於細胞的影響 95
3-12 生物複合材料抑制小鼠乳腺癌細胞（4T1）於生物體內的生長 97
3-13 於生物體內驗證脂多醣誘導細胞焦亡反應的發生 104
4 第四章結論及未來展望 107
參考文獻 108
附錄 114

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指導教授

謝發坤(Fa-Kuen Shieh)

審核日期

2024-8-19

推文