類沸石咪唑骨架材料封裝大腸桿菌誘發癌症細胞細胞焦亡之研究

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王鉅霖(Ju-Lin Wang) 查詢紙本館藏

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論文名稱

類沸石咪唑骨架材料封裝大腸桿菌誘發癌症細胞細胞焦亡之研究
(Induction of Pyroptosis in Cancer Cells Using Escherichia Coli Encapsulation into Zeolitic Imidazole Frameworks)

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摘要(中)

癌症又稱為惡性腫瘤，傳統常見的癌症治療方如手術移除、放射治療
以及化學治療，但經常伴隨著嚴重的副作用例如抗藥性或是正常細胞也會
一併清除。細菌腫瘤療法(bacteria-mediated cancer therapy)提供癌症治療另外
一種群新的選擇，
但目前細菌癌症療法依舊存在一些困境，如何於常規滅菌方式下維持細
菌的活性，同時避免細菌外膜上的脂多醣(Lipopolysaccharide, LPS)外露而影
響輸送的精確性，造成患者的副作用。因此學者們致力於有機和無機材料結
合生物體的生物復合材料，進而改善細菌對於人體環境的適應性。
金屬有機骨架材料（Metal-Organic Frameworks, MOFs）具有相當的多樣
性，因此近年來常被應用在結合生物系統相關的研究。本研究利用類沸石咪
唑骨架材料-90（Zeolitic Imidazolate Frameworks, ZIF-90）包覆大腸桿菌
(Escherichia coli, E. coli)表面，形成單一晶體包覆大腸桿菌(E. coli@ZIF-90)
進行了多種癌症細胞療法相關的體外細胞實驗。實驗結果發現將 E. coli 以
單晶形式包覆在 ZIF-90 中，能夠防止 E. coli 外膜上的 LPS 暴露在巨噬細胞
中，有效降低巨噬細胞的免疫反應，表示 ZIF-90 作為藥物載體將能更安全
的在人體中輸送大腸桿菌。此外，在比較多種癌症細胞株後，發現 E.
coli@ZIF-90 對乳腺癌細胞具有 53 %毒殺效果證實 E. coli@ZIF-90 能夠誘
發乳腺癌細胞的發炎反應，並造成癌症細胞凋亡。由細胞中鋅離子濃度的差
ii
異，發現癌症細胞比正常細胞對 ZIF-90 吞噬能力較強，證明 E. coli@ZIF90 對乳腺癌細胞有較高專一性。最後透過檢測乳腺癌細胞凋亡過程觀察發
炎因子生成量，發現 E. coli 是透過 ZIF-90 送入細胞內，其外膜上的 LPS 引
發癌症細胞焦亡(Pyroptosis)的發生，造成癌細胞死亡。
本研究證實利用類沸石咪唑骨架材料作為藥物載體能更有效的將細菌
送入癌症細胞內部，封裝的細菌可以在腫瘤細胞酸性環境下釋放，露出細菌
外膜上的脂多醣，以此誘發細胞發炎並進一步導致細胞焦亡。此研究結果提
供細菌做為癌症療法的新方式，這類輸送的方式可以有效將且專一將細菌
釋放到癌細胞內部，不會對正常細胞產生傷害。這些實驗結果將有助於更進
一步地應用於癌症治療領域，提供更加有效和安全的治療方案。相信本研究
成果能為癌症治療帶來更多的突破。

摘要(英)

Cancer, a leading cause of death worldwide, is traditionally treated with
surgery, radiation therapy, and chemotherapy, which can have adverse effects. To
overcome these limitations, bacteria-mediated cancer therapy targets cancer cells
selectively using bacteria and their toxins. This approach aims to minimize
damage to healthy tissue while activating the immune system against cancer cells.
However, bacterial cancer therapy faces challenges such as deactivation during
conventional sterilization methods and obstacles in delivering bacteria to tumor
locations due to the body′s immune response. Additionally, the presence of
lipopolysaccharide (LPS) on the outer membrane of bacteria can trigger immune
responses and lead to side effects. Thus, how to improve the adaptability of
bacteria to the human body is still a challenge.
In this study, we showcased the encapsulation of E. coli bacteria within
Zeolitic Imidazolate Framework-90 (ZIF-90), a subclass of Metal-Organic
Frameworks (MOFs). This encapsulation process enhances the bacteria′s
protection against lysozyme and antibiotics within the human body. In vitro
experiments demonstrated the successful delivery of E. coli into cancer cells using
ZIF-90 particles, resulting in reduced immune responses from macrophages and
specific targeting of breast cancer cells. Furthermore, the 53% toxicity effect of
E. coli@ZIF-90 on breast cancer cells confirmed that E. coli@ZIF-90 induced
inflammatory responses in breast cancer cells and caused apoptosis of cancer cells.
Moreover, the measurement of zinc ion concentration revealed the selective
targeting of cancer cells by E. coli@ZIF-90 while avoiding damage to normal
cells. Additionally, the study investigated the mechanism of intracellular
pyroptosis induced by E. coli, attributing it to the effects of LPS in cancer cells.
iv
This research provides a promising approach to cancer therapy by effectively
delivering bacteria into cancer cells and inducing inflammation and apoptosis.
The findings contribute to the development of more effective and safer treatment
options in the field of cancer therapy. It is expected that further advancements in
this research will lead to breakthroughs in cancer treatment.

關鍵字(中)

★ 細菌癌症療法
★ 類沸石咪唑骨架材料
★ 脂多醣
★ 乳腺癌細胞
★ 細胞焦亡

關鍵字(英)

★ : bacteria-mediated cancer therapy
★ Zeolitic Imidazolate Framework
★ lipopolysaccharide
★ breast cancer cells
★ pyroptosis

論文目次

中文摘要 i
Abstract iii
目錄 v
圖目錄 ix
表目錄 xi
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
1-2-1 癌症簡述 6
1-2-2 乳腺癌簡述 6
1-2-3 傳統常見癌症療法 7
1-3 細菌癌症療法 8
1-3-1 細菌癌症療法 8
1-3-2 細菌癌症療法問題 10
1-3-3 大腸桿菌(Escherichia coli, E. coli) 10
1-3-4 脂多醣(Lipopolysaccharide, LPS) 11
1-3-5 藥物載體 13
1-4 癌症細胞株和正常細胞株 14
1-4-1 胃腺癌細胞株（AGS） 14
1-4-2 鼻咽癌細胞（NPC-TW01） 14
1-4-3 肺癌細胞（A549） 14
1-4-4 鱗狀細胞癌（A431） 15
1-4-5 腎癌細胞（A498） 15
1-4-6 小鼠乳腺癌細胞(4T1) 15
1-4-7 人類乳腺癌細胞(Hs 578T) 16
1-4-8 人類正常乳腺上皮細胞(H184) 16
1-5 細胞死亡 17
1-5-1 細胞凋亡(Apoptosis) 17
1-5-2 細胞焦亡(Pyroptosis) 17
1-6 研究動機與目的 19
第二章實驗部分 22
2-1 實驗藥品與設備 22
2-2 實驗儀器原理 26
2-2-1 Ｘ射線粉末繞射儀 (Powder X-ray Diffractometer, PXRD) . 26
2-2-2 螢光顯微鏡 27
2-2-3 紫外/可見光光譜儀 28
2-2-4 酵素連結免疫吸附測定法(Enzyme-linked immunosorbent assay) 28
2-2-5 西方墨點法(Western Blotting) 29
2-2-6 細胞存活率測試 31
2-3 實驗步驟 32
2-3-1 大腸桿菌之培養步驟 34
2-3-2 合成類沸石咪唑骨架材料-90 封裝大腸桿菌 35
2-3-3 細胞培養 36
2-3-4 酵素連結免疫吸附測定法(Enzyme-linked immunosorbent assay) 36
2-3-5 西方墨點法(Western Blotting) 37
第三章結果與討論 41
3-1 大腸桿菌生物復合材料之相關鑑定 41
3-2 E. coli@ZIF-90 對多種癌症細胞株之存活率測試 44
3-3 E. coli@ZIF-90 誘發發炎反應測試 47
3-4 ClearColi@ZIF-90 之相關鑑定 50
3-5 乳腺癌細胞發炎反應測試 51
3-6 正常細胞之細胞存活率以及發炎反應測試 53
3-7 細胞對類沸石咪唑骨架材料-90 之吞噬能力測試 54
3-8 Lipopolysaccharide 所誘發之細胞焦亡驗證實驗 56
3-9 抑制 Gasdermin D 之細胞存活率測試 62
3-10 正常細胞中細胞焦亡反應 64
第四章結論及未來展望 67
第五章參考文獻 68

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指導教授

謝發坤

審核日期

2023-8-14

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