臺灣博碩士論文加值系統

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

1.Chiono, V. and C. Tonda-Turo, Trends in the design of nerve guidance channels in peripheral nerve tissue engineering. Progress in Neurobiology, 2015. 131: p. 87-104.
2.Subramanian, A., U.M. Krishnan, and S. Sethuraman, Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration. Journal of Biomedical Science, 2009. 16(1): p. 108.
3.Gu, X., et al., Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Progress in Neurobiology, 2011. 93(2): p. 204-230.
4.Cuvillon, P., et al., The Continuous Femoral Nerve Block Catheter for Postoperative Analgesia: Bacterial Colonization, Infectious Rate and Adverse Effects. Anesthesia & Analgesia, 2001. 93(4).
5.Granato, A.E.C., et al., Polypyrrole increases branching and neurite extension by Neuro2A cells on PBAT ultrathin fibers. Nanomedicine: Nanotechnology, Biology and Medicine, 2018. 14(6): p. 1753-1763.
6.Sangsanoh, P., et al., Enhancement of biocompatibility on aligned electrospun poly (3‐hydroxybutyrate) scaffold immobilized with laminin towards murine neuroblastoma Neuro2a cell line and rat brain‐derived neural stem cells (mNSCs). Polymers for Advanced Technologies, 2018. 29(7): p. 2050-2063.
7.Pisani, S., et al., Biocompatible polymeric electrospun matrices: Micro–nanotopography effect on cell behavior. Journal of Applied Polymer Science, 2020: p. 49223.
8.陳柏瑋, 以快速原型法和靜電紡絲製備幾丁聚醣/果膠之雙層複合支架用於骨軟骨組織修復之研究, in 化學工程與生物科技系化學工程碩士班. 2019, 國立臺北科技大學: 台北市. p. 88.
9.林慧宣, 以3D列印製作海藻酸鈉水凝膠和幾丁聚醣/聚乙烯醇奈米纖維雙層複合支架用於組織修復, in 化學工程研究所. 2016, 國立臺北科技大學: 台北市. p. 0.
10.劉子豪, 含噬菌體之幾丁聚醣薄膜用於皮膚傷口抑菌之研究, in 化學工程與生物科技系化學工程碩士班. 2019, 國立臺北科技大學: 台北市. p. 91.
11.Naveed, M., et al., Chitosan oligosaccharide (COS): An overview. International Journal of Biological Macromolecules, 2019. 129: p. 827-843.
12.Molnar, C. and J. Gair, 16.1 Neurons and Glial Cells. Concepts of Biology-1st Canadian Edition, 2013.
13.Martini, R., J. Groh, and U. Bartsch, Comparative biology of Schwann cells and oligodendrocytes. The biology of oligodendrocytes. Cambridge University Press, Cambridge, 2010: p. 19-48.
14.Mahar, M. and V. Cavalli, Intrinsic mechanisms of neuronal axon regeneration. Nature Reviews Neuroscience, 2018. 19(6): p. 323-337.
15.Yiu, G. and Z. He, Glial inhibition of CNS axon regeneration. Nature Reviews Neuroscience, 2006. 7(8): p. 617-627.
16.Avellino, A.M., et al., Differential macrophage responses in the peripheral and central nervous system during wallerian degeneration of axons. Experimental neurology, 1995. 136(2): p. 183-198.
17.Schmidt, C.E. and J.B. Leach, Neural tissue engineering: strategies for repair and regeneration. Annual review of biomedical engineering, 2003. 5(1): p. 293-347.
18.Belanger, K., et al., Recent strategies in tissue engineering for guided peripheral nerve regeneration. Macromolecular bioscience, 2016. 16(4): p. 472-481.
19.Gaudet, A.D., P.G. Popovich, and M.S. Ramer, Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. Journal of neuroinflammation, 2011. 8(1): p. 110.
20.Gu, X., F. Ding, and D.F. Williams, Neural tissue engineering options for peripheral nerve regeneration. Biomaterials, 2014. 35(24): p. 6143-6156.
21.Kaikabo, A.A., S.M. AbdulKarim, and F. Abas, Evaluation of the efficacy of chitosan nanoparticles loaded ΦKAZ14 bacteriophage in the biological control of colibacillosis in chickens. Poultry Science, 2017. 96(2): p. 295-302.
22.Ohkawa, K., et al., Electrospinning of chitosan. Macromolecular rapid communications, 2004. 25(18): p. 1600-1605.
23.Sabaghi, M., et al., Active edible coating from chitosan incorporating green tea extract as an antioxidant and antifungal on fresh walnut kernel. Postharvest Biology and Technology, 2015. 110: p. 224-228.
24.Chang, Y.H., et al., Removal of Hg2+ from aqueous solution using alginate gel containing chitosan. Journal of applied polymer science, 2007. 104(5): p. 2896-2905.
25.Abdelsattar, A.S., et al., Encapsulation of E. coli phage ZCEC5 in chitosan–alginate beads as a delivery system in phage therapy. AMB Express, 2019. 9(1): p. 87.
26.Muanprasat, C. and V. Chatsudthipong, Chitosan oligosaccharide: Biological activities and potential therapeutic applications. Pharmacology & Therapeutics, 2017. 170: p. 80-97.
27.Liu, X., et al., Multidentate zwitterionic chitosan oligosaccharide modified gold nanoparticles: stability, biocompatibility and cell interactions. Nanoscale, 2013. 5(9): p. 3982-3991.
28.Yang, Y., et al., Effect of chitooligosaccharide on neuronal differentiation of PC-12 cells. Cell biology international, 2009. 33(3): p. 352-356.
29.Chae, S.Y., M.-K. Jang, and J.-W. Nah, Influence of molecular weight on oral absorption of water soluble chitosans. Journal of Controlled Release, 2005. 102(2): p. 383-394.
30.Lian, Z., et al., EDTA-functionalized magnetic chitosan oligosaccharide and carboxymethyl cellulose nanocomposite: Synthesis, characterization, and Pb(II) adsorption performance. International Journal of Biological Macromolecules, 2020. 165: p. 591-600.
31.Zou, P., et al., Advances in characterisation and biological activities of chitosan and chitosan oligosaccharides. Food Chemistry, 2016. 190: p. 1174-1181.
32.Hu, H., et al., Glutaraldehyde–chitosan and poly (vinyl alcohol) blends, and fluorescence of their nano-silica composite films. Carbohydrate Polymers, 2013. 91(1): p. 305-313.
33.Miyamoto, D.M., et al., Chitosan Microspheres loaded with holmium-165 produced by Spray Dryer for liver cancer therapy: preliminary experiments. 2011.
34.Baldino, L., et al., Complete glutaraldehyde elimination during chitosan hydrogel drying by SC-CO2 processing. The Journal of Supercritical Fluids, 2015. 103: p. 70-76.
35.Park, J.H., et al., Electrospinning and characterization of poly(vinyl alcohol)/chitosan oligosaccharide/clay nanocomposite nanofibers in aqueous solutions. Colloid and Polymer Science, 2009. 287(8): p. 943-950.
36.Alhosseini, S.N., et al., Synthesis and characterization of electrospun polyvinyl alcohol nanofibrous scaffolds modified by blending with chitosan for neural tissue engineering. International journal of nanomedicine, 2012. 7: p. 25-34.
37.Li, C., et al., Silver nanoparticle/chitosan oligosaccharide/poly(vinyl alcohol) nanofibers as wound dressings: a preclinical study. International journal of nanomedicine, 2013. 8: p. 4131-4145.
38.Quek, S.Y., J. Hadi, and H. Tanambell, Application of electrospinning as bioactive delivery system. 2019.
39.Li, Z. and C. Wang, Effects of Working Parameters on Electrospinning, in One-Dimensional nanostructures: Electrospinning Technique and Unique Nanofibers. 2013, Springer Berlin Heidelberg: Berlin, Heidelberg. p. 15-28.
40.Nitti, P., et al., Influence of Nanofiber Orientation on Morphological and Mechanical Properties of Electrospun Chitosan Mats. Journal of Healthcare Engineering, 2018. 2018: p. 3651480.
41.Xu, C.Y., et al., Aligned biodegradable nanofibrous structure: a potential scaffold for blood vessel engineering. Biomaterials, 2004. 25(5): p. 877-886.
42.McGrath, S. and D.v. Sinderen, Bacteriophage: genetics and molecular biology. 2007: Caister Academic Press.
43.Labrie, S.J., J.E. Samson, and S. Moineau, Bacteriophage resistance mechanisms. Nature Reviews Microbiology, 2010. 8(5): p. 317-327.
44.Richter, Ł., et al., Recent advances in bacteriophage-based methods for bacteria detection. Drug Discovery Today, 2018. 23(2): p. 448-455.
45.Monk, A., et al., Bacteriophage applications: where are we now? Letters in applied microbiology, 2010. 51(4): p. 363-369.
46.Kakasis, A. and G. Panitsa, Bacteriophage therapy as an alternative treatment for human infections. A comprehensive review. International Journal of Antimicrobial Agents, 2019. 53(1): p. 16-21.
47.Doss, J., et al., A review of phage therapy against bacterial pathogens of aquatic and terrestrial organisms. Viruses, 2017. 9(3): p. 50.
48.沈涵榆, 以3D列印法製作含噬菌體之纖維狀褐藻酸鈉水凝膠敷料, in 化學工程與生物科技系化學工程碩士班. 2019, 國立臺北科技大學: 台北市. p. 96.
49.Kumar, M. and A. Katyal, Data on retinoic acid and reduced serum concentration induced differentiation of Neuro-2a neuroblastoma cells. Data in Brief, 2018. 21: p. 2435-2440.
50.Jia, Y.-T., et al., Fabrication and characterization of poly (vinyl alcohol)/chitosan blend nanofibers produced by electrospinning method. Carbohydrate polymers, 2007. 67(3): p. 403-409.
51.Buchko, C.J., et al., Processing and microstructural characterization of porous biocompatible protein polymer thin films. Polymer, 1999. 40(26): p. 7397-7407.
52.Lee, H.W., et al., Poly (vinyl alcohol)/chitosan oligosaccharide blend submicrometer fibers prepared from aqueous solutions by the electrospinning method. Journal of applied polymer science, 2009. 111(1): p. 132-140.
53.Zhang, C., et al., Study on morphology of electrospun poly (vinyl alcohol) mats. European polymer journal, 2005. 41(3): p. 423-432.
54.Koosha, M. and H. Mirzadeh, Electrospinning, mechanical properties, and cell behavior study of chitosan/PVA nanofibers. Journal of Biomedical Materials Research Part A, 2015. 103(9): p. 3081-3093.
55.Han, S.O., et al., Electrospinning of cellulose acetate nanofibers using a mixed solvent of acetic acid/water: Effects of solvent composition on the fiber diameter. Materials Letters, 2008. 62(4-5): p. 759-762.
56.Koski, A., K. Yim, and S. Shivkumar, Effect of molecular weight on fibrous PVA produced by electrospinning. Materials Letters, 2004. 58(3-4): p. 493-497.
57.Çay, A., M. Miraftab, and E. Perrin Akçakoca Kumbasar, Characterization and swelling performance of physically stabilized electrospun poly(vinyl alcohol)/chitosan nanofibres. European Polymer Journal, 2014. 61: p. 253-262.
58.Zamre, K., et al., Chemical cross-linking of chitosan/polyvinyl alcohol electrospun nanofibers. Materiali in tehnologije, 2016. 50(5): p. 663-666.
59.Aronino, R., et al., Removal of viruses from surface water and secondary effluents by sand filtration. Water research, 2009. 43(1): p. 87-96.
60.YAMASHITA, M., et al., Effect of Alcohols on Escherichia coil Phages. Biocontrol Science, 2000. 5(1): p. 9-16.
61.Ackermann, H.-W., Frequency of morphological phage descriptions in the year 2000. Archives of virology, 2001. 146(5): p. 843-857.
62.Habeeb, A. and R. Hiramoto, Reaction of proteins with glutaraldehyde. Archives of biochemistry and biophysics, 1968. 126(1): p. 16-26.
63.Handa, H., et al., Recognition of Salmonella typhimurium by immobilized phage P22 monolayers. Surface Science, 2008. 602(7): p. 1392-1400.
64.Dalmasso, M., et al., Three new Escherichia coli phages from the human gut show promising potential for phage therapy. PloS one, 2016. 11(6): p. e0156773.
65.Nogueira, F., et al., Immobilization of bacteriophage in wound-dressing nanostructure. Nanomedicine: Nanotechnology, Biology and Medicine, 2017. 13(8): p. 2475-2484.
66.Bahrami, S. and M. Nouri, Chitosan-poly (vinyl alcohol) blend nanofibers: morphology, biological and antimicrobial properties. e-Polymers, 2009. 9(1).
67.Bhattacharjee, A.S., et al., Bacteriophage therapy for membrane biofouling in membrane bioreactors and antibiotic‐resistant bacterial biofilms. Biotechnology and Bioengineering, 2015. 112(8): p. 1644-1654.
68.Cheng, W., et al., Incorporation of bacteriophages in polycaprolactone/collagen fibers for antibacterial hemostatic dual‐function. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2018. 106(7): p. 2588-2595.
69.Merzlyak, A., S. Indrakanti, and S.-W. Lee, Genetically engineered nanofiber-like viruses for tissue regenerating materials. Nano letters, 2009. 9(2): p. 846-852.
70.Zha, F., et al., Effects of surface condition of conductive electrospun nanofiber mats on cell behavior for nerve tissue engineering. Materials Science and Engineering: C, 2020: p. 111795.
71.Lee, J.Y., W.-J. Chung, and G. Kim, A mechanically improved virus-based hybrid scaffold for bone tissue regeneration. RSC advances, 2016. 6(60): p. 55022-55032.
72.Christopherson, G.T., H. Song, and H.-Q. Mao, The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials, 2009. 30(4): p. 556-564.
73.Salehi, M., et al., Regeneration of sciatic nerve crush injury by a hydroxyapatite nanoparticle-containing collagen type I hydrogel. The Journal of Physiological Sciences, 2018. 68(5): p. 579-587.
74.Xu, C., et al., Biodegradable and electroconductive poly(3,4-ethylenedioxythiophene)/carboxymethyl chitosan hydrogels for neural tissue engineering. Materials Science and Engineering: C, 2018. 84: p. 32-43.