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研究生:李哲宇
研究生(外文):Jhe-yu Li
論文名稱:明膠/藍藻蛋白複合膜的製備,材料特性與皮膚纖維母細胞生長之探討
論文名稱(外文):Preparation, characterization and skin fibroblast growth studies of gelatin/cyanophycin composite scaffolds.
指導教授:曾文祺曾文祺引用關係
指導教授(外文):Wen-chi Tseng
口試委員:曾文祺
口試委員(外文):Wen-chi Tseng
口試日期:2013-07-18
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
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:2013
畢業學年度:101
語文別:中文
論文頁數:74
中文關鍵詞:戊二醛明膠藍藻蛋白皮膚纖維母細胞
外文關鍵詞:glutaraldehydegelatincyanophycinskin fibroblast
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  藍藻蛋白(Cyanophycin granule polypeptide)是一種非核醣體合成蛋白質,其結構是由天門冬氨酸(aspartic acid)為主鏈,精氨酸(arginine)與離氨酸(lysine)為側鏈所組成的聚合物;而明膠是由部分水解性的天然膠原蛋白所組成,主要為一重複連續的甘氨酸(glycine)-脯氨酸(proline)-基脯氨酸(hydroxiproline)序列及其他胺基酸所構成的分子。
  明膠與藍藻蛋白具有良好的生物相容性,但由於藍藻蛋白其彎曲強度和韌性較差,限制了藍藻蛋白的應用範圍,而明膠具有親水性(Hydrophilic)良好、成膜性佳與顯著的機械性能(mechanical)等優點,故本研究目的是以戊二醛(glutaraldehyde)進行交聯反應製備不同比例的明膠/藍藻蛋白複合膜,期望能增強其材料的機械性質及提高皮膚纖維母細胞的生長。
  本實驗將明膠與藍藻蛋白共混成溶液後進行交聯反應,以製備成不同比例之複合膜,隨後以掃描式電子顯微鏡(scanning electron microscope, SEM)觀察複合膜其表面和切面結構,並使用材料試驗機進行機械性質的量測,與熱重差分析法(thermogravimetric analysis, TGA)對複合膜的裂解點進行測試,且包含了含水率(swelling)及接觸角的測量;並使用皮膚纖維母細胞在不同比例複合膜上進行培養,以MTT法測試其相對存活率。
  實驗結果顯示,在SEM的材料表面觀察與接觸角測量中,觀察到複合膜隨著藍藻蛋白的所占含量的提高,孔洞與層狀結構更臻明顯;而在機械性質的測試中看出,適量的材料共混有助於提升其抗拉強度;從TGA量測結果發現,使用水溶性藍藻蛋白成膜的組別,會有兩段的裂解溫度表現,推測是水溶性藍藻蛋白成膜的組別交聯反應較佳,升溫過程中交叉鏈接的部分會被先行裂解,而產生此現象。在吸水率方面,看出複合膜會隨明膠所占比例越少,吸水率隨之減少。在明膠/藍藻蛋白複合膜經72小時的皮膚纖維母細胞培養後,可得知其材料有助於細胞的生長。
  從上述結果可歸納出,適量的藍藻蛋白與明膠共混有助於提升其抗拉強度,可擴大藍藻蛋白的應用範圍,並提高纖維母細胞的生長。
  Cyanophycin granule polypeptide (CGP) is a non-ribosomal synthesis protein. The polymer consists of an aspartic acid backbone with arginine and lysine as side chains. Gelatin is composed of partial hydrolysis of collagen, and is the product of almost continuous repeats of the glycine- proline -hydroxyproline sequence.
  Gelatin and CGP have good biocompatibility. However, the strength and toughness of CGP is poor, which limits its applications. The gelatin has good hydrophilicity, film forming and mechanical properties. In this study, we prepared different ratios of gelatin/CGP composite scaffolds by cross-linking reaction with glutaraldehyde in an effort to improve the mechanical properties of the composite material and the growth of skin fibroblast.
  We mixed different ratios of gelatin and CGP in solution and the mixture was cross-linked with glutaraldehyde. We used scanning electron microscopy (SEM) to observe the surface, cross-section and cell attachment morphology of the composite scaffolds. A universal test machine was used to detect the tensile strength and elongation of the material. The decomposition temperatures were measured by thermogravimetric analysis(TGA), and the hydrophilicity was examined by a contact angle meter. The growth of skin fibroblast was monitored by MTT assay.
  The results showed that the pore and layer structures became more evident with an increasing percentage of CGP. The mechanical test showed that the mixture of CGP and gelatin can improve the tensile strength at an optimal ratio of gelatin and CGP . The composite of soluble CGP cross-linked with gelatin showed two decomposition temperatures by TGA analysis. The composites also provided a better cell growth environment for skin fibroblast on the gelatin/CGP composite scaffolds.
  In conclusion, the composite of CGP cross-linked gelatin can improve the tensile strength and the growth of fibroblasts which extend the applications of CGP.
中文摘要 I
Abstract III
誌 謝 V
目錄 VI
圖目錄 X
表目錄 XIII
第一章 緒論 1
1.1 前言 1
1.2 研究動機 2
第二章 文獻回顧 3
2.1 明膠 3
2.1.1 明膠介紹 3
2.1.2 明膠特性 3
2.1.3 明膠結構 4
2.1.4 明膠的提取 6
2.1.5 凝膠強度(Bloom value) 7
2.1.6 等電點(isoelectric point) 8
2.1.7 明膠的應用 8
2.2 藍藻蛋白(cyanophycin) 10
2.2.1 藍藻蛋白簡介 10
2.2.2 藍藻蛋白結構 10
2.2.3 藍藻蛋白的生產 11
2.2.4 藍藻蛋白的應用 13
第三章 實驗 14
3.1 實驗材料 14
3.1.1菌株 14
3.1.2 細胞株 14
3.1.3 細胞培養基 14
3.1.4 抗生素 14
3.2 實驗藥品 15
3.3 藥品配製 17
3.4 實驗器材 19
3.5 實驗設備 20
3.6 實驗流程 21
3.7 實驗步驟 21
3.7.1 微生物培養 21
3.7.2 自菌體純化藍藻蛋白 23
3.7.3 培養皮膚纖維母細胞Hs68 25
3.7.4 製備藍藻蛋白/明膠複合膜 26
3.7.5 明膠/藍藻蛋白複合膜之特性分析 31
3.7.6動物細胞的生長觀察及毒性測試 35
第四章 結果與討論 43
4.1 明膠/藍藻蛋白複合膜分析 43
4.1.1 巨觀觀察明膠/藍藻蛋白複合膜 43
4.1.2以9,10-phenanthrenequinone分析明膠/藍藻蛋白複合膜 43
4.1.3 以掃描式電子顯微鏡觀察明膠/藍藻蛋白複合膜表面型態 44
4.1.4以掃描式電子顯微鏡觀察明膠/藍藻蛋白複合膜切面結構 44
4.2明膠/藍藻蛋白複合膜之特性分析 45
4.2.1明膠/藍藻蛋白複合膜之機械性質 45
4.2.2熱重差掃描卡量測明膠/藍藻蛋白複合膜 46
4.2.3明膠/非水溶性藍藻蛋白複合膜之含水率測量 47
4.2.4明膠/藍藻蛋白複合膜之親疏水性 47
4.3 Hs68 cell於明膠/藍藻蛋白複合膜之生長情形 48
4.3.1 Hs68 cell於明膠/藍藻蛋白複合膜上之毒性測試 48
4.3.2 Hs68 cell於明膠/藍藻蛋白複合膜上之蛋白質含量測試 48
4.3.3 Hs68 cell於明膠/藍藻蛋白複合膜上之DNA含量測試 49
4.3.4觀察Hs68 cells於明膠/藍藻蛋白複合膜上貼附情形 49
第五章 結論 51
圖表 52
參考文獻 71
1. Yeh, M.K., et al., A novel cell support membrane for skin tissue engineering: Gelatin film cross-linked with 2-chloro-1-methylpyridinium iodide. Polymer, 2011. 52: p. 996-1003.
2. Scuder, N., et al., Clinical Application of Autologous Three-cellular Cultured Skin Substitutes Based on Esterified Hyaluronic Acid Scaffold: Our Experience. in vivo 2009. 23: p. 991-1004.
3. Metcalfe, A.D. and M.W.J. Ferguson, Bioengineering skin using mechanisms of regeneration and repair. Biomaterials 2007. 28: p. 5100-5113.
4. Hrabchak, C., L. Flynn, and K.A. Woodhouse, Biological skin substitutes for wound cover and closure. Expert Rev Med Devices, 2006. 3: p. 373-85.
5. Mano, J.F., et al., Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends. J. R. Soc. Interface, 2007.
6. Bigi, A., S. Panzavolta, and K. Rubini, Relationship between triple-helix content and mechanical properties of gelatin films. Biomaterials 2004. 25: p. 5675-5680.
7. Ulubayram, K., et al., EGF containing gelatin-based wound dressings. Biomaterials, 2001. 22: p. 1345-1356.
8. Schrieber, R. and H. Gareis, Gelatin Handbook : Theroy and Industrial Practice. 2007, London: Wiley.
9. Djabourov, M., J. Leblond, and P. Papon, Gelation of aqueous gelatin solutions. I. Structural investigation. Journal de Physique, 1988. 49 p. 319-332
10. Friess, W., Collagen-biomaterial for drug delivery. European Journal of Pharmaceutics and Biopharmaceutics 1998. 45: p. 113-136.
11. Gomez-Guillen, M.C., et al., Functional and bioactive properties of collagen and gelatin from alternative sources: A review. Food Hydrocolloids 2011. 25: p. 1813-1827.
12. 連成東, 明膠的特性與銀鹽膠片的保存條件. 檔案學通訊, 1999. 4: p. 46~48.
13. Rose, P.I., Gelatine in Encyclopedia of Polymer Science and Engineering. Vol. 7. 1987, London: Wiley. 488-513.
14. Gross, J., Comparative biochemistry of collagen. Vol. 5. 1963, New York: Academic Press.
15. Djabourov, M., J.P. Lechaire, and F. Gaill, Structure and rheology of gelatin and collagen gels. Biorheology, 1993. 30: p. 191-205.
16. Montero, P., et al., Characterization of Hake (Merluccius merluccius L.) and Trout (Salmo irideus Gibb) Collagen J. Agric. Food Chem, 1990. 38: p. 694-699.
17. Standard Methods for the Sampling and Testing of Gelatins, Gelatin Manufacturers Institute ofAmerica, ed. fifth. 1986, New York.
18. Gomez-Guillen, M.C., et al., Fish gelatin: a renewable material for the development of active biodegradable films. Trends in Food Science and Technology, 2009. 20: p. 3-16.
19. Veis, A., The macromolecular chemistry of gelatin. Academic Press, 1964.
20. Ledward, D.A., Gelation of gelatins. Functional Properties of food macromolecules, 1986: p. 171-201.
21. Ledward, D.A., Gelatin. Hanbook of hydrocolloids, 2000: p. 67-86.
22. Borzi, A., Le comunicazioni intracellulari delle Nostochinee. Malpighia, 1887: p. 74-203.
23. Obst, M., et al., Isolation and Characterization of Gram-Positive Cyanophycin-Degrading BacteriasKinetic Studies on Cyanophycin Depolymerase Activity in Aerobic Bacteria. Biomacromolecules, 2004. 5: p. 153-161.
24. Krehenbrink, M., F.B. Oppermann-Sanio, and A. Steinbuchel, Evaluation of non-cyanobacterial genome sequences for occurrence of genes encoding proteins homologous to cyanophycin synthetase and cloning of an active cyanophycin synthetase from Acinetobacter sp. strain DSM 587. Archives of Microbiology, 2002. 177: p. 371-380.
25. Tseng, W.C., et al., Expression of Synechocystis sp. PCC6803 cyanophycin synthetase in Lactococcus lactis nisin-controlled gene expression system (NICE) and cyanophycin production. Biochemical Engineering Journal 2013.
26. Dembinskat, M.E. and M.M. Allen, Cyanophycin Granule Size Variation in Aphanocapsa. Journal of General Microbiology 1988. 134: p. 295-298.
27. Mooibroek, H., et al., Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production. Appl Microbiol Biotechnol 2007(257-267).
28. Simon, R.D., Cyanophycin Granules from the Blue-Green Alga Anabaena cylindrica: A Reserve Material Consisting of Copolymers of Aspartic Acid and Arginine. Proceedings of the National Academy of Sciences, 1971. 68: p. 265-267.
29. Aboulmagd, E., F.B. Oppermann-Sanio, and A. Steinbuchel, Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308. Archives of Microbiology, 2000. 174: p. 297-306.
30. Hai, T., F.B. Oppermann-Sanio, and A. Steinbuchel, Purification and characterization of cyanophycin and cyanophycin synthetase from the thermophilic Synechococcus sp. MA19. FEMS Microbiology Letters, 1999. 181: p. 229-236.
31. Steinle, A., K. Bergander, and A. Steinbuchel, Metabolic Engineering of Saccharomyces cerevisiae for Production of Novel Cyanophycins with an Extended Range of Constituent Amino Acids. Applied and Environmental Microbiology, 2009. 75: p. 3437-3446.
32. Martin Obst, M. and A. Steinbuchel, Microbial Degradation of Poly(amino acid)s. Biomacromolecules 2004. 5: p. 1166-1176.
33. Neubauer, K., et al., Isolation of cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression. Journal of Biotechnology 2012. 158: p. 50-58.
34. Hai, T., F.B. Fred Bernd Oppermann-Sanio, and A. Steinbu‥chel, Molecular Characterization of a Thermostable Cyanophycin Synthetase from the Thermophilic Cyanobacterium Synechococcus sp. Strain MA19 and In Vitro Synthesis of Cyanophycin and Related Polyamides. Applied and Environmental Microbiology, 2002: p. 93-101.
35. Obst, M. and A. Steinbuchel, cyanophycin-an Ideal Bacterial Nitrogen Storage Material with Unique Chemical Properties. Microbiol Monogr, 2006: p. 168-187.
36. Khor, E., Review : Methods for the treatment of collagenous tissues for bioprostheses. Biomaterials 1997. 18: p. 95-105.
37. Migneault, I., et al., Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. BioTechniques, 2004. 37: p. 790-802.
38. Imani, R., M. Rafienia, and S.H. Emami, Synthesis and characterization of glutaraldehyde-based crosslinked gelatin as a local hemostat sponge in surgery: An in vitro study. Bio-Medical Materials and Engineering, 2013. 23: p. 211-224.
39. Magun, B.E. and J.W. Kelly, A new fluorescent method with phenanthrenequinone for the histochemical demonstration of arginine residues in tissues Journal of Histochemistry and Cytochemistry, 1969. 17.
40. Lam, C.N.C., et al., Study of the advancing and receding contact angles: liquid sorption as a cause of contact angle hysteresis. Advances in Colloid and Interface Science, 2002. 96: p. 169-191.
41. Stockert, J.C., et al., MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. Acta Histochemica, 2012. 114: p. 785-796.
42. Deiber, J.A., et al., Characterization of cross-linked polyampholytic gelatin hydrogels through the rubber elasticity and thermodynamic swelling theories. Polymer 2009. 50: p. 6065-6075.
43. Bradford, M.M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 1976. 72: p. 248-254.
44. Okochi, M., et al., Fluorometric observation of viable and dead adhering diatoms using TO-PRO-1 iodide and its application to the estimation of electrochemical treatment. Appl Microbiol Biotechnol 1999. 51: p. 364-369.
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