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研究生:江旻擇
研究生(外文):CHIANG, MING-TSE
論文名稱:以水溶性幾丁聚醣製作奈米絲薄膜後表面固定噬菌體用於抑菌及神經修復之研究
論文名稱(外文):Bacteriophage Immobilization on Electrospun Water-Soluble Chitosan for Bacteriostasis and Nerve Repair
指導教授:林忻怡林忻怡引用關係
指導教授(外文):LIN, HSIN-YI
口試委員:林忻怡鄭國忠王大銘
口試委員(外文):LIN, HSIN-YICHENG, KUO-CHUNGWANG, DA-MING
口試日期:2021-01-18
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:化學工程與生物科技系化學工程碩士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:84
中文關鍵詞:噬菌體抑菌敷料幾丁聚醣靜電紡絲神經細胞
外文關鍵詞:BacteriophageBacteriostatic dressingElectrospinningNerve cellChitosan
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  • 被引用被引用:1
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組織工程已成為修復神經的途徑之一,靜電紡絲所形成的奈米和次微米纖維的支架模擬天然的細胞外基質可以提供良好的環境幫助細胞再生。隨著抗生素的濫用細菌耐藥性的提升,使抗生素無法達到預期的效果,因此噬菌體療法逐漸受到重視。本實驗將噬菌體以化學方式交聯於順向幾丁聚醣奈米絲上,製成有抑菌功能及幫助神經修復的支架。本實驗以大腸桿菌(DH5α)作為宿主與海水共培養,從中分離出能抑制宿主的噬菌體,使用靜電紡絲的方式將水溶性幾丁聚醣製成支架,以戊二醛化學交聯加強其結構,再將噬菌體與戊二醛以化學交聯的方式固定於材料表面,製造含有噬菌體之幾丁聚醣的電紡薄膜。首先進行製作先以不同濃度之幾丁聚醣/聚乙烯醇溶液電紡絲測試其能否成有奈米纖維結構,並進行物理性質測試、體外細胞測試以探討含噬菌體之幾丁聚醣奈米絲對於抑菌及神經修復之影響。首先利用掃描式電子顯微鏡(SEM)觀察不同濃度的幾丁聚醣/聚乙烯醇電紡絲,以確定電紡薄膜含有纖維狀結構,實驗觀察結果10%幾丁聚醣/13%聚乙烯醇的濃度下有明確的纖維狀結構。再進行電紡上的噬菌體活性測試、抑菌測試,以確定電紡薄膜上的噬菌體經過實驗處理依然維持其活性與抑菌功能,噬菌體表面固定數量測試結果電紡薄膜表面固定1.4x107 PFU噬菌體,抗菌測試結果顯示含有噬菌體的電紡薄膜菌落數有明顯的低於不含噬菌體的靜電紡絲(p<0.05),以掃描式電子顯微鏡觀測靜電紡薄膜的抑菌情況,結果顯示在含噬菌體組的電紡薄膜上有明顯的降低。接著進行體外細胞測試以探討含噬菌體固定於電紡薄膜表面對於抑菌及神經修復之影響。在細胞測試的部分,電紡薄膜上DNA定量測試顯示有含噬菌體組的細胞量含量明顯高於不含噬菌體組(p<0.05),含噬菌體的電紡紡薄膜有更好的生長速度。最後在不同時間電紡纖維結構的降解型態結果顯示,表面固定噬菌體的電紡薄膜不會影響電紡薄膜含有的奈米纖維之降解速度。本實驗結果得知,噬菌體固定於電紡薄膜表面仍然可以保持其活性及抑菌能力,且不會影響電紡薄膜奈米纖維的降解速度,對於細胞有良好的生長情形,綜合以上結果含有噬菌體的電紡絲在抑菌及神經修復工程具有應用上的潛力。
Tissue engineering has become one of the ways to repair nerves. The nano- and sub-micron fiber scaffolds formed by electrospinning mimic the natural extracellular matrix and can provide a good environment to help cell regeneration. In this experiment, the phages were chemically cross-linked on the chitin excised nanowires to make a scaffold with antibacterial function and help nerve repair. In this experiment, antibiotics (DH5α) were used as the host and co-cultured with seawater, and phages that could inhibit the host were isolated from it. Electrospinning was used to gradually replace chitin into the scaffold, and glutaraldehyde chemical cross-linking strengthened its structure. The phage and glutaraldehyde are grafted on the surface of the material in a chemical cross-linking manner to produce an electrospun film containing chitosan ester of the phage. First of all, it is made to test whether it can be formed into a nanofiber structure by electrospinning with different concentrations of chitosan/polyvinyl alcohol solution, and conduct physical property test, and in vitro cell test to explore the effect of bacteriophage-containing tetrandine The effect of antibacterial and nerve repair. First, the scanning electron microscope (SEM) was used to observe the electrospinning of different concentrations of chibutyl/polyvinyl alcohol, and the multi-layer fibrous structure of the electrospun film was used. The experimental observation result was 10% chibutyl/13% polyvinyl alcohol. Carry out electrospinning phage activity test and antibacterial test. The phage on the electrospun membrane still maintains its activity and antibacterial function through experimental treatment. The results of the phage graft test result is electrospinning grafted 1.4x107 PFU phage, antibacterial test The results showed that the number of colonies of electrospun film containing phage significantly exceeded the number of electrospinning that excludes phage (p<0.05). Scanning electron microscope was used to observe the bacteriostasis of the electrospun film. There is a significant reduction on the spun film. Then an in vitro cell test was carried out to explore the effect of grafting the phage-containing phage onto the electrospun film on bacteriostasis and nerve repair. In the cell test part, the DNA quantitative test on the electrospun film showed that the cell content of the phage-containing group was significantly higher than that of the non-phage group (p<0.05), and the electrospun film containing phage had a better growth rate. Finally, the results of the degradation patterns of the electrospun fiber structure at different times show that the electrospinning of the grafted phage does not affect the degradation rate of the nanofibers contained in the electrospun film. The results of this experiment know that the phage grafted on the electrospun film can still maintain its activity and antibacterial ability, and will not affect the degradation rate of the electrospun film nanofiber, and has a good growth mechanism for the cells. The above results contain phage Electrospinning has application potential in antibacterial and nerve repair engineering.
摘要 I
ABSTRACT III
誌謝 V
目錄 VI
圖目錄 X
表目錄 XII
第一章 緒論 1
1.1 前言 1
1.2 研究目的 1
第二章文獻回顧 2
2.1 神經構造及傷口修復 2
2.1.1神經構造 2
2.1.2傷口癒合 3
2.2神經組織工程 4
2.3幾丁聚醣 5
2.3.1幾丁聚醣介紹及性質 5
2.3.2交聯方式 6
2.4聚乙烯醇 7
2.5靜電紡絲 8
2.6噬菌體 9
2.6.1 噬菌體介紹及性質 9
第三章實驗方法與材料 11
3.1 實驗材料 11
3.2 實驗方法 12
3.2.1 實驗設計 12
3.2.2 實驗架構 13
3.2.3 分離海水中的噬菌體及培養 14
3.2.3.1 大腸桿菌(DH5α)培養 14
3.2.3.2 噬菌體培養 15
3.2.3.3噬菌體外觀(TEM) 19
3.2.4 靜電紡絲薄膜製備及參數設定 20
3.2.4.1 電紡溶液配製 20
3.2.4.2 電紡設備架設 21
3.2.4.3 交聯 22
3.2.4.4電紡薄膜滅菌 22
3.2.5電紡參數測試(SEM) 23
3.2.6 製備表面固定噬菌體的電紡薄膜 23
3.2.7 噬菌體測試 25
3.2.7.1 甘胺酸水溶液對噬菌體的影響 25
3.2.7.2以戊二醛固定噬菌體於電紡薄膜表面上的噬菌體數量 26
3.2.8 微生物測試 28
3.2.8.1 不同濃度噬菌體溶液抑菌測試 28
3.2.8.2噬菌體固定於電紡薄膜表面之抑菌測試 29
3.2.8.3以掃描式電子顯微鏡觀測大腸桿菌分佈 31
3.2.9 生物相容性測試 31
3.2.9.1 細胞培養 31
3.2.9.2細胞接種於電紡薄膜上 33
3.2.9.3 電紡薄膜上神經母細胞DNA定量 33
3.2.9.5短時間內電紡薄膜神經細胞細胞貼附 35
3.2.9.6 電紡薄膜上細胞型態(掃描式電子顯微鏡,SEM) 36
3.2.9.7 電紡薄膜上分化後細胞型態(掃描式電子顯微鏡,SEM) 36
3.2.10 物理測試 37
3.2.10.1 電紡直徑分析 37
3.2.10.2 不同時間電紡纖維降解型態 37
3.2.10.3 接觸角測試 38
3.3統計分析 39
第四章實驗結果與討論 40
4.1電紡參數測試(SEM) 40
4.2物理測試 45
4.2.1 電紡薄膜外觀 45
4.2.2 電紡直徑分析 46
4.2.3 不同時間電紡纖維結構的降解型態 48
4.2.4 接觸角測試 51
4.3噬菌體測試 52
4.3.1 噬菌體外觀(TEM) 52
4.3.2 甘胺酸水溶液對噬菌體活性影響測試 53
4.3.3 以戊二醛固定噬菌體於電紡薄膜表面上的表面固定數量 54
4.4微生物測試 57
4.4.1 不同濃度噬菌體溶液抑菌測試 57
4.4.2 噬菌體固定於電紡薄膜表面之抑菌測試 58
4.4.3 以掃描式電子顯微鏡觀測大腸桿菌生長 60
4.5細胞毒性測試 62
4.5.1 電紡薄膜上神經母細胞DNA定量 62
4.5.2 短時間內電紡薄膜上神經細胞貼附 64
4.5.3 電紡薄膜上細胞型態(cell morphology by SEM) 66
4.5.4 電紡薄膜上分化後細胞型態(SEM) 69
第五章結論 73
參考文獻 74
附錄一、藥品配置 79
附錄二、DNA定量標準曲線 84

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