類沸石咪唑骨架材料封裝大腸桿菌對未來癌症療法應用之前期研究

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蔡承樺(Cheng-Hua Tsai) 查詢紙本館藏

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類沸石咪唑骨架材料封裝大腸桿菌對未來癌症療法應用之前期研究

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至系統瀏覽論文 (2027-7-1以後開放)

摘要(中)

微生物在生活中被廣泛應用，例如汙水處理使用各類微生物來分解有機廢物或是重金屬，利用厭氧菌來製造醇類，醫學上也有利用細菌進行治療的細菌癌症療法。但不可避免的在作用環境中總有不利於微生物生存的條件存在，像是水中可能含有抗菌劑、厭氧菌作用環境無法完全阻隔氧氣、病人體內的溶菌酶和抗生素等，都會減弱微生物的活性及存活能力。因此科學家便致力於研究有機或無機材料結合生物體的生物複合材料，來增強生物細胞對於環境壓力的適應性。
金屬有機骨架材料 (MOFs)，可藉由調整金屬離子與有機配體，根據需求調整其孔洞性質、比表面積、化學穩定性等，具有相當的多樣性，因此近期常被應用於與生物系統結合之研究。
本研究藉由將類沸石咪唑骨架材料-90 (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)，以及將類沸石咪唑骨架材料-8 (ZIF-8) 合成於大腸桿菌表面形成塗層 (E. coli⊂ZIF-8)的產物，並於高溫環境中來探討E. coli@ZIF-90與E. coli⊂ZIF-8兩者不同包覆情況對提高大腸桿菌耐熱能力的影響。同時也以這兩種生物複合材料進行了與細胞癌症療法相關的體外細胞實驗：透過誘發巨噬細胞活化生成而得的多種細胞激素(Cytokines)，探討這兩種生物複合材料於誘發細胞免疫反應的多寡；以及將E. coli@ZIF-90與不同pH值環境下的巨噬細胞一同培養，觀測材料是否有崩解現象及大腸桿菌釋放後與細胞是否有作用力，探討ZIF-90材料作為大腸桿菌載體的潛力。以期待未來將本研究成果應用於癌症治療之可行性以及在癌症治療領域帶來更進一步的突破。

摘要(英)

Microorganisms are widely used in life. For example, decomposing organic waste in sewage, producing alcohol by anaerobic fermentation, besides, bacteria can also be used in cancer treatment called “bacteria cancer therapy”. However, there are always some factors which will threaten their survival. Antiseptics exist in water, oxygen exist in the media of anaerobic bacteria, lysozyme and antibiotics in patients. These conditions all make microorganisms deactivate. Therefore, scientists research on combining organic or inorganic materials with cells in order to enhance their adaptability toward environmental stress.
Metal organic frameworks (MOFs) have variable pore properties, specific surface area, chemical stability. Scientists can adjust these properties by changing the precursors to fit their purpose. Therefore, the development of biocomposites by combinig Metal-organic frameworks and living systems is rapidly emerging.
In this study, we successfully synthesized two kinds of biocomposites, Escherichia coli (E. coli) cells was encapsulated into single crystal zeolitic imidazolate framework-90 (ZIF-90) or been coating by zeolitic imidazolate framework-8 (ZIF-8). After these combinations, E. coli gains the ability to survive the treatment of heat. E. coli can still regrowth after removing the material. At the same time, we studied whether these E. coli biocomposites will determine the activation of macrophages. The induced expression of inflammatory cytokines was evaluated using a mouse inflammation antibody array. E. coli@ZIF-90 significantly prevented the release of inflammatory mediators from E. coli compared to free E. coli and E. coli⊂ZIF-8.
On the other hand, ZIF-90 also had the incredible performance on being a delivery carrier. We found out the bacteria phagocytosis was observed though the incubation with E. coli@ZIF-90 in pH 6.0 culture media, simulated the environments of the cancer cells compared to E. coli@ZIF-90 were no decomposed in pH 7.0 media. This study may pave the new way in the history of bacterial cancer therapy and hope this study will be the “carrier” in bacterial cancer therapy.

關鍵字(中)

★ 類沸石咪唑骨架材料
★ 大腸桿菌
★ 細菌免疫療法
★ 微生物封裝

關鍵字(英)

★ E. coli
★ ZIF-90
★ ZIF-8

論文目次

目錄
中文摘要 i
Abstract ii
目錄 iv
圖目錄 vii
表目錄 viii
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 3
1-1-3 類沸石咪唑骨架材料-8/-90 5
1-2 微生物 6
1-2-1 微生物 6
1-2-2 大腸桿菌 (Escherichia coli, E. coli) 6
1-2-3 質體 (Plasmid) 7
1-2-4 細菌癌症療法 (Bacterial Cancer Therapy) 8
1-3 研究動機與目的 11
第二章實驗 13
2-1 實驗藥品及材料 13
2-2 實驗儀器 15
2-2-1 場發掃描式電子顯微鏡 (Field-emission Scanning Electron Microscope, FE-SEM) 16
2-2-2 X射線粉末繞射儀 (Power X-ray Diffractometer, PXRD) 17
2-2-4 攜帶式分光光度計 (Ultrospec 10 cell Density Meter) 18
2-2-5 螢光顯微鏡 (Fluorescence Microscopy) 18
2-3 實驗使用之酵素 20
2-3-1 溶菌酶 (Lysozyme) 20
2-3-2 蛋白酶 (Protease) 20
2-4 實驗步驟 21
2-4-1 大腸桿菌之培養步驟 21
2-4-2 奈米級類沸石咪唑骨架材料-8塗層之大腸桿菌 (E. coli⊂ZIF-8) 之合成步驟 22
2-4-3 微米級類沸石咪唑骨架材料-90封裝之大腸桿菌 (E. coli@ZIF-90) 之合成步驟 22
2-4-4 類沸石咪唑骨架材料-8/-90封裝大腸桿菌 (E. coli⊂ZIF-8/E. coli@ZIF-90) 抵禦抗生素之能力測試實驗步驟 23
2-4-5 類沸石咪唑骨架材料-90封裝大腸桿菌 (E. coli@ZIF-90) 耐熱能力測試之實驗步驟 23
2-4-6 類沸石咪唑骨架材料-8封裝大腸桿菌 (E. coli⊂ZIF-8) 耐熱能力測試之實驗步驟 24
2-4-7 大腸桿菌 (E. coli) 耐熱能力測試之實驗步驟 24
2-4-8 單批微米級類沸石咪唑骨架材料-90封裝之大腸桿菌 (E. coli@ZIF-90)含有之活體E.coli數量測定 24
2-4-9 奈米級類沸石咪唑骨架材料-90合成步驟 25
2-4-10 奈米級類沸石咪唑骨架材料-90塗層之大腸桿菌 (E. coli⊂nano ZIF-90) 之合成步驟 25
2-4-11 奈米級類沸石咪唑骨架材料-90塗層之大腸桿菌再次結晶實驗之相關步驟 26
2-4-12 奈米級類沸石咪唑骨架材料-90包覆順鉑 (Cisplatin@nano ZIF-90) 之合成步驟 26
2-4-13 奈米級類沸石咪唑骨架材料-90包覆順鉑塗層之大腸桿菌 (E. coli ⊂ Cisplatin@nano ZIF-90) 之合成步驟 26
2-4-14 奈米級類沸石咪唑骨架材料-90包覆順鉑塗層之大腸桿菌再次結晶實驗之相關步驟 27
第三章結果與討論 28
3-1 大腸桿菌生物複合材料之相關鑑定 28
3-1-1 X射線粉末繞射儀的鑑定結果 28
3-1-2 場發掃描式電子顯微鏡之成像結果 31
3-1-3 奈米級類沸石咪唑骨架材料-8塗層於大腸桿菌/微米級類沸石咪唑骨架材料-90封裝大腸桿菌 (E. coli⊂ZIF-8/E. coli@ZIF-90) 之抗生素耐受活性測試 33
3-1-4 奈米級類沸石咪唑骨架材料-8塗層於大腸桿菌/微米級類沸石咪唑骨架材料-90封裝大腸桿菌 (E. coli⊂ZIF-8/E. coli@ZIF-90) 之耐熱能力測試 35
3-1-5 奈米級類沸石咪唑骨架材料-8塗層於大腸桿菌/微米級類沸石咪唑骨架材料-90封裝大腸桿菌 (E. coli⊂ZIF-8/E. coli@ZIF-90) 發炎反應測試 36
3-1-6微米級類沸石咪唑骨架材料-90封裝大腸桿菌 (E. coli@ZIF-90) 與巨噬細胞 Raw 264.7之吞噬作用測試 38
第四章結論 40
參考文獻 41

圖目錄
圖 1- 1常見之MOFs的方式17 2
圖 1- 2高通量合成 2
圖 1- 3 (1) 類沸石咪唑骨架材料 ; (2) 天然沸石 3
圖 1- 4 Yaghi團隊於2008年發表之ZIFs結構 4
圖 1- 5 大腸桿菌示意圖 7
圖 1- 6 pET-28a 質體示意圖38 8
圖 1- 7 細菌癌症療法示意圖55 10

圖 2- 1 掃描式電子顯微鏡構造示意圖56 16
圖 2- 2 布拉格定律38 17
圖 2- 3 螢光顯微鏡之光路示意圖57 19

圖 3- 1 E. coli⊂ZIF-8、E. coli@ZIF-90之PXRD圖 28
圖 3- 2 nano ZIF-90之PXRD圖 29
圖 3- 3 E. coli⊂nano ZIF-90之PXRD圖 29
圖 3- 4 透過再次結晶所得之E. coli@ZIF-90之PXRD圖 30
圖 3- 5 E. coli@ZIF-90之掃描式電子顯微鏡影像 31
圖 3- 6 E. coli⊂ZIF-8之掃描式電子顯微鏡影像 32
圖 3- 7 E. coli⊂nano ZIF-90之掃描式電子顯微鏡影像 33
圖 3- 8 大腸桿菌於不同封裝方式下氨苄青黴素耐受性之測試 34
圖 3- 9 大腸桿菌於60度環境下耐熱能力之測試 35
圖 3- 10 大腸桿菌於不同狀況下誘發各種細胞激素生成之相對濃度長條圖 37
圖 3- 11大腸桿菌於不同狀況下誘發(a)IL-6及(b)TNF-α生成之濃度長條圖 37
圖 3- 12 E. coli@ZIF-90於不同環境中培養釋放大腸桿菌誘發巨噬細胞的吞噬作用之螢光顯微鏡影像 39

表目錄
表 2- 1 實驗藥品及材料 13
表 2- 2 實驗儀器 15

參考文獻

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指導教授

謝發坤(Fa-Kuen Shieh)

審核日期

2022-8-18

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