臺灣博碩士論文加值系統

本篇論文的研究方向主要著重於膠原蛋白結合甲殼素之複合材料對於抑菌與促進細胞增生活性的研究。膠原蛋白是結締組織的主要成分由三股螺旋結構而成的生物高分子，本研究以老鼠膠原蛋白第一型(Rat collagen type I )為材料，利用胃蛋白酵素（Pepsin）在弱酸中進行水解，使膠原蛋白在水溶液之溶解度增加。本研究使用的幾丁聚醣材料包括：酸溶性去乙醯度91.78%、酸溶性去乙醯度75.37%、水溶性去乙醯度56.55%、水溶性去乙醯度75.75%之幾丁聚醣。研究結果發現酸溶性幾丁聚醣溶液具有極佳的抗菌效果，水溶性的幾丁聚醣抑菌效果較差，並且隨著幾丁聚醣的去乙醯度越高，抑菌效果越好。另外透過化學修飾之表面帶負電水溶性幾丁聚醣具抗菌效果非常差，這反映出幾丁聚醣抑菌的機制和表面的正電荷有關。本研究也對於幾丁聚醣的抗菌的機制做初步的鑑定。在抑菌濃度範圍內，發現隨著細菌量的增多，實驗樣品中的DNA溢出量也跟著增多，因此推估，幾丁聚醣可能是藉由破壞細菌的細胞壁而達到抑制細菌生長的效果。依據上述研究結果製備出幾丁聚醣與膠原蛋白複合材料並探討其活性，研究發現膠原蛋白加入幾丁聚醣不會影響抗菌的效果，並且此複合材料仍可以促進細胞增生。

The study was prepared composite material containing collagen and chitosan, and investigated its antimicrobial activity and its effect on proliferation of fibroblasts. Collagen is the major component of connective tissue and it has triple helix structure. The type I collagen isolated from rat tail and hydrolyzed with pepsin was used in this study. Chitosan has been reported to have antimicrobial activity in previous study. We tested antimicrobial activity of four different kinds of chitosan such as acid-soluble chitosan (deacetylation 91.78%), acid-soluble chitosan(deacetylation 75.37%), water-soluble chitosan(deacetylation 56.55%) and water-soluble chitosan(deacetylation 75.75%). The result showed that the acid-soluble chitosan has good antimicrobial activity but the water-soluble chitosan doesn’t. Chitosan with higher degree of deacetylation showed better antimicrobial activity. However, when the positive charge of chitosan was changed to negative charge, it lost its antimicrobial activity. This result indicated that the positive charge of chitosan is critical to its antimicrobial activity. We also found that chitosan induced DNA leakage from E. coli in quantitative manner and it suggested that chitosan inhibited the growth of E.coli by attacking and lysed its cell wall. In this study, we prepared collagen and chitosan composite material and examined its effect on cell proliferation & antimicrobial activities. We found the composite material with good antimicrobial and cell proliferation activities.

中文摘要 ………………………………………………………………I
英文摘要 ………………………………………………………………Ⅱ
目錄 ……………………………………………………………………Ⅲ
圖目錄 …………………………………………………………………Ⅶ
表目錄 …………………………………………………………………XV



第一章 序論 1

第二章 膠原蛋白的製備與應用
一、膠原蛋白簡介 3
二、膠原蛋白在生物科技上之應用與淺力 10
三、實驗方法
第一部分 材料 21
第二部分 儀器 22
第三部分 實驗方法 23
1. 膠原蛋白容易配製處理 23
2. 羥基脯胺酸定量 23
3. 膠原蛋白之溶解度測試 24
4. 不同溫度對胃蛋白酵素水解膠原蛋白的影響 24
5. IR定性 25
6. DSC定性 25
四、結果與討論 26
五、結論 35

第三章 幾丁聚醣抗菌效果
一、幾丁聚醣簡介 36
二、幾丁聚醣之淺力與應用 50
三、實驗方法
第一部分 材料 54
第二部分 儀器 55
第三部分 實驗方法
1.IR定性 56
2.幾丁聚醣去乙醯度測試 56
3.黏度計 57
4.XRD定性 58
5.DSC定性 58
6.MIC抗菌之濃度 58
7.不織布抗菌效果 59
8.甲殼素不同濃度與相同時間之抗菌效果 60
9.甲殼素相同濃度與不同菌量之抗菌效果 60
10.Genomic DNA製備法看菌DNA溢出量 60
11.0.1%甲殼素的抑菌速度 61
12.SEM之觀察 61
四、結果與討論 69
五、結論 97

第四章 膠原蛋白與甲殼素的製備與應用
一、研究動機 98
二、膠原蛋白與甲殼素之應用 98
三、實驗方法
第一部分 材料 100
第二部分 儀器 101
第三部分 實驗方法
1.DSC測定 102
2.不織布抗菌效果 103
3.甲殼素不同濃度與相同時間之抗菌效果 103
4.甲殼素相同濃度與不同菌量之抗菌效果 104
5.Genomic DNA製備法看菌DNA溢出量 104
6.0.1%甲殼素的抑菌速度 105
7.細胞培養 105
8.SEM之觀察 105
四、結果與討論 107
五、結論 131

第五章 總結 132
參考文獻 133

[1]願其胜 蔣麗霞，膠原蛋白與臨床醫學 第二軍醫大學出版(2003)
[2]修復醫學與組織工程 化學工業出版社(2003)
[3]黃彥富 湯正明 徐善慧 揭開膠原蛋白神秘面紗 科學發展（2003）
[4]島野紘一，如何認識膠原蛋白，國際村文庫書店有限公司，台灣(1996)
[5]將晃榮，膠原蛋白的驚人療效，世茂出版社，台灣（2002）
[6]Lubert, S.“Biochemistry 4th Edition”W. H. Freeman and Company, New York, p.31-32
[7]Karl, E. K. et al. “ Collagen fibril formation” Biochem. J.316, 1-11(1996)
[8]Anderson,S. M. L. et al.“Sircol Assay:A method for quantification of collagen”Biochem.Soc.Trans.19,48-54(1991)
[9]Kesava ,G. ,R.et.al“A Simplified Method for the Analysis of Hydroxyproline in Biological Tissues”Clinical Biochemistry,29,225-229(1996)
[10]Konrad, B. et al. “Supercoild Protein Motifs :The Collagen Triple –Helix and the a-Helical Coil” Journal of structure biology,122,17-29 (1998)
[11]Tarnowski et al. “Effects of treatment protocols and subcutaneous implantation on bovine pericardium: a Raman spectroscopy study” Journal of Biomedical Optics,8,179-184(2003)
[12]Friess,W. “Basic thermoanalytical studies of insoluble collagen matrices” Biomaterials, 17, 2289-2294(1996)
[13]Pietrucha ,K. “Changes in denaturation and rheological properties of collagen–hyaluronic acid scaffolds as a result of temperature dependencies”International Journal of Biological Macromolecules,36,299-304(2005)
[14]Kobayashi, H.“Development of a new in vitro chemosensitivity test using collagen gel droplet embedded culture and image analysis for clinical usefulness”Recent Results in Cancer Research,161,48-61(2003)
[15] Komsa, P. , R. et al. “Modification of Loery’s Mothed for collagen concentration measurement ”J.Biochem.Biophys.Methods 320,33-43(1996)
[16] Spradling, K. et al. “Epsin 3 is a novel extracellualar matrix induced transcript specific to wounded epithelia” J. Bol. Chem,276 ,29257-29267 (2001)
[17]Pieper, J.S. et al. “Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate” Biomaterials ,20 , 847- 858 (1999)
[18]江晃榮，幾丁質、幾丁聚醣的發現與研發，世茂出版社，34-47，1998。
[19]鄭家昇、吳俊毅、李昌憲，基質對幾丁聚醣發酵之影響，第五屆生化工程研討會,p269-272(2000)
[20]徐薇如,民國89 年,”幾丁聚醣/藻酸鈣生物高分子對重金屬吸附行為之研究”,國立交通大學應用化學研究所碩士論文.
21.黃啟嘉，醫用微生物學(上冊)，合記圖書出版社，55-65，1991。
[22]陳建宏、張孝全、林千惠，抗菌及抗M.R.S.A. 纖維之發展現況，化工資訊，59-64，2000。
[23]Chung,Y C. et al. “Relationship between antibacterial activity of chitosan and surface characteristics of cell wall” Acta. Pharmaco.l Sin., 25 , 932-936 (2004)
[24]Wu, T. et al. “Physicochemical properties and bioactivity of fungal chitin and chitosan ” J. Agric Food Chem. 53, 3888-3894 (2005)
[25]JUNG, B. O. et al.“Preparation of Amphiphilic Chitosan and Their Antimicrobial Activities”Journal of Applied Polymer Science, 72,1713-1719 (1999)
[26]Cho, Y. W. et al. “Preparation and solubility in acid and water of partially deacetylated chitins"Biomacromolecules, 1, 609-614 (2000)
[27]Kato,Y. et al. “ Biological characteristics of lactosaminated N-succinyl-chitosan as a liver-specific drug carrier in mice”Journal of Controlled Release,70,295-309 (2001)
[28]Boenig ,H. V. et al, Plasma Science and Technology, Cornell University Press (1982)
[29]Jia ,Z., shen ,D., Xu ,W., “ Synthesis and Antibacterial Activities of QuaternarynAmmonium Salt of Chitosan” Carbonhydrate Research, Vol. 333, 1-6 (2001)
[30]Li ,Z., Zhuang ,X. P., Liu ,X. F., Guan ,Y. L., Yao ,K. D.“ Study on Antibacterial o-Carboxymethylated Chitosan /Cellulose Blend Film from LiCl/N,NDimethylacetamide Solution” Journal of Applied Polymer Science, Vol.43,1541-1547 (2002).
[31]Sugano et al.“In-plane quasi-isotropic organic superconductor di[bis(ethylenedithiolo)tetrathiafulvalene] bis(isothiocyanato) cuprate (I), (BEDT-TTF) 2[Cu(NCS)2]: Polarized reflectance spectra”American Physical Society,Vol.37,9100-9102(1988) .
[32]Sugano et al.“Theoretical Studies of a Light-Induced Change in Multiplet Satellites of 3p-Photoelectron Spectra of Transition-Metal Compounds”, American Physical Society,Vol.44,1527-1530 (1980) .
[33]Jennings et al.“Surface barrier for electrons in metals” American Physical Society,Vol.37, 6113–6120 (1988)
[34]Lindsay et al.“Observation of Low-Lying Raman Bands in DNA by Tandem Interferometry” American Physical Society,Vol. 53, 1853–1855 (1984)
[35]Kono et al."Surface and bulk core-level shifts of the Si(111)√3 √3 -Ag surface: Evidence for a charged √3 √3 layer” American Physical Society,Vol. 58, 1555–1558 (1987)
[36]Muzzarelli et al.“Antimicrobial Properties of N-Carboxybutyl Chitosan” ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,Vol.34, 2019-2023(1990)
[37]Kendra, D. F. and Hadwiger, L. A. “Characterization of the smallest chitosanoligomer that is maximally antifungal to Fusarium soloni and elicits pisatinformation in Pisum sativum. ”Exp. Mycol.,8,276-281 (1984).
[38]Chung YC et al." Relationship between antibacterial activity of chitosan and surface characteristics of cell wall”Acta Pharmacol Sin. ,7, 932-936 (2004).
[39]Tanaka et al.“Step Permeability and the Relaxation of Biperiodic Gratings on Si(001)” American Physical Society,Vol. 78, 3342–3345 (1997).
[40]Shigemasa,Y. et al, Minami, S. et al. “Chitosan-inducing Hemorrhagic Pneumonia in Dogs”Carbohydr. Polym., Vol. 29, 241-246,(1996).
[41]Maeda et al.“Magnetic-field penetration depth and the lower critical field of the quasi-two-dimensional superconductor Bi2Sr2CaCu2Oy” American Physical Society, 46, 14234–14237 (1992).
[42]Matsumoto et al.“Competition between the singlet-spin liquid state and the magnetic ground state in a two-chain spin-½ antiferromagnetic ladder compound LaCuO2.5: A 63Cu NMR study” American Physical Society, Vol.53, R11942–R11945 (1996) .
[43]Tanigawa et al.“Upper critical field and anisotropy of the high-Tc Bi2Sr2Ca2Cu3Ox phase” American Physical Society,Vol. 45, 7414–7417 (1992) .
[44]Shibata, Y., Foster, L.A., Metzger, W.J., Quentin, N.M“Alveolar Macrophage priming by intravenous administration of chitin particles, polymers of N-acetyl-D-glucosamine” in mice. Infect. and Immun., 65, 1734-1741(1997).
[45]Peluso, G., Petillo, O., Ranieri, M., Santin, M., Ambrosio, L., Calabro, D., Avallone, B., Balsamo, “G.Chitosan-mediated stimulation of macrophage function” Biomaterials, 15, 1215-1220(1994).
[46]Iwama, G., and Nakanishi, T.. “Fish Physiology Volume XV: The fish immune system”.Academic Press,15,( 1996)
[47]Kim SK, Park PJ, Yang HP, Han SS. “ Subacute toxicity of chitosan oligosaccharide inSprague-Dawley rats. Arzneimittelforschung. ” 51,769-74(2001)
[48]Knapczyk J. Krówczynski L. Pawlik B. Liber Z. “Pharmaceutical dosage forms with chitosan. In: Skjåk-Bræek G. Anthonsen T. Sandford P. (Eds.), Chitin and Chitosan: Sources, Chemistry, Biochemistry, Physical Properties and Applications. Elsevier Applied Science. London. 665-670 (1989)
[49]Arai K. Kinumaki T. Fujita T. “Toxicity of chitosan. ”Bull. Tokai Reg. Fish. Res.,43,:89-94 (1968)
[50]Deuchi K. Kanauchi O. Shizukuishi M. Kobayashi E. “ Continuous and massive intake of chitosan affects mineral and fat-soluble vitamin status in rats fed on a high-fat diet” Biosci Biotechnol Biochem,59,1211-1216 (1995)
[51]Jing SB. Li L. Ji D. Takiguchi Y. Yamaguchi T. “Effect of chitosan on renal function in patients with chronic renal failure. ” J Pharm Pharmacol. ,49, 721-723 (1997)
[52]Wuolijoki E. Hirvela T. Ylitalo P. “Decrease in serum LDL cholesterol with microcrystalline chitosan. ” Methods Find Exp Clin Pharmacol. , 21, 357-361 (1999)
[53]Pittler MH. Abbot NC. Harkness EF. Ernst E. “Randomized, double-blind trial of chitosan for body weight reduction. ” Eur J Clin Nutr. 53 ,379-381 (1999)
[54]Cho YW, Cho YN, Chung SH, Yoo G, Ko SW.“ Water-soluble chitin as a wound healing accelerator” Biomaterials ,20, 2139-2145 (1999) .
[55]Vande, V.et al. “Evaluation of the biocompatibility of a chitosan scaffold in mice” J Biomed. , 59 , 585-590 (2002)
[56]Chung , W.Y.et al, “Properties of the poly(vinyl alcohol)/chitosan blend and its effect on the culture of fibroblast in vitro” Biomaterials , 20 , 1479-1487 (1999)
[57]Ylitalo R. Lehtinen S. Wuolijoki E. Ylitalo P. Lehtimaki T. “Cholesterol-lowering properties and safety of chitosan. ”Arzneimittelforschung. ,52 ,1-7 (2002)
[58]Sabnis,S. and Block ,L. H. “Improved infrared spectrotion method for the analysis of degree N-deacetylation of chitosan” Polym Bull,39,67-71,1997
[59]Khan, T. A . et al. “Reorting degree of deacetylation values of chitosan : the influence of analytical methods”J Pharm Pharmaceut Sci,5,205-212(2002)
[60]Ghobad G. Maghami,George A. F. Roberts “Evaluation of the viscomertic constants for chitosan” Makromol.Chem., 189, 195-200 (1988)
[61]Zhang Y. et al. “Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction” Carbohydrate research ,340 , 1914-1917 (2005)
[62]Ren, D. et al. “The enzymatic degradation and swelling properties of chitosan matrices with different degrees of N-acetylation” Carbohydrate research , 340, 2403-2410 (2005)
[63]Ritthidej , G. C. et al. “Moist heat treatment on physicochemical change of chitosan salt films” International Journal of Pharmaceutics ,232,11-22(2002)
[64]Gibaly,I. E. et al. “Development and in vitro evaluation of novel floating chitosan microcapsules for oral use: comparison with non-floating chitosan microspheres” International Journal of Pharmaceutics,249,7-21,(2002)
[65]Gerasimenko ,D. V. et al. “Antibacterial Effects of Water-Soluble Low-Molecular-Weight Chitosans on Different Microorganisms”Applied Biochemistry and Microbiologycal,40,301-306
[66]Dau, G. et al. “Bactreicidal and Morphological Effects of NE-2001,a Novel Synthetic Agent Directed against Helicobacter polyri” Antimicrob. Agents Chemother,49,3468-3473,2005
[67]Qi, L. et al. “Preparation and antibacterial activity of chitosan nanoparticles”Carbohydrate Research,339,2693-2700(2004)
[68]Gavini , E. et al.“Mucoadhesive vaginal tablets as veterinary delivery system for the controlled release of an antimicrobial drug,acriflavine”AAPS PharmSci,3,1-7(2002)
[69]Xie, et al. “Preparation of Water-Soluble Chitosan Derivatives and Their Antibacterial Activity” Journal of Applied Polymer Science,85,1357-1361(2002)
[70]Xie W. , Xu P., Wang W., Lin Q., “Preparation and Antibacterial Activity of a Water-Soluble Chitosan Derivative”Carbohydrate Polymers, 50, 35-40, (2002)
[71]Benesch J., Tengvall P. “Blood Protein Adsorption onto Chitosan”, Biomaterials 23, 2561-2568, (2002)
[72]Silva , C. C. et al.“On the piezoelectricity of collagen – chitosan films” Phys. Chem. Chem. Phys.,3,4154-4157(2001)
[73]Domsazy, J.G. et al. “Evaluation of infrared spectroscopic techniques for analyzing chitosan” Makromol. Chem. ,186 ,1671-1677 (1985)
[74]Sannan ,T. te al. “Studies on chitin: 7. I.r. spectroscopic determination of degree of deacetylation” Polymer. , 19 , 458-459 (1978)
[75]Ren, D. te al. , “The enzymatic degradation and swelling properties of chitosan matrices with different degrees of N-acetylation” Carbohydrate Research , 340, 2403-2410 (2005) .
[76]Zhang, Y.et al . “Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction” Carbohydrate Research , 340 , 1914-1917 (2005) .
[77]林明哲、廖峻德、林尚明、簡世峰、黃毓瑾、宋銘志
，去乙醯度和pH值對幾丁聚醣抗菌活性之研究。
[78]Amaral,I. F. et al “Rat bon marrow stromal cell ostegenic differentiation and fibronectin adsorption on chitosan membranes : T The effect of the degree of acetylation” RODOC, 3, 387-397(2005)
[79]羅宗軒、陳明庭、呂旭彥，新鮮冰凍豬皮之臨床實驗，中華重建整外醫誌，5，34~38，1996
[80]王存福、王先震，表淺性傷口之合成皮膚帶用品治療，中華重建整外醫誌，3卷， 4期，219~222 (1994)
[81]王先震、湯有銘、周林興，合成性生物敷料Biobrane之沾黏性動物實驗，中華重建整外醫誌，1卷，1號，48~51（81）