研究生: |
洪健哲 Chien-Che Hung |
---|---|
論文名稱: |
藍藻蛋白接枝透明質酸製備多孔性支架 Cyanophycin grafted hyaluronic acid to prepare porous 3D scaffold |
指導教授: |
曾文祺
Wen-Chi Tseng |
口試委員: |
何郡軒
Jinn-Hsuan Ho 方翠筠 Tsuei-Yun Fang |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 化學工程系 Department of Chemical Engineering |
論文出版年: | 2015 |
畢業學年度: | 103 |
語文別: | 中文 |
論文頁數: | 49 |
中文關鍵詞: | 藍藻蛋白 、透明質酸 、立體孔洞支架 |
外文關鍵詞: | cyanophycin, hyaluronic acid, porous 3D scaffold |
相關次數: | 點閱:544 下載:2 |
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藍藻蛋白(cyanophycin)是一種非核醣體合成的蛋白質,於西元1887年由Borzi在顯微鏡下觀察藍綠藻菌時首次發現。
由於以藍綠藻生產藍藻蛋白的產率偏低,因此我們將大腸桿菌進行基因改質,使其含有藍藻蛋白合成酶的基因,並以IPTG or 乳糖進行誘導,藉此來提升藍藻蛋白的產率,天然的藍藻蛋白是以天門冬胺酸 (aspartic acid, Asp) 與精胺酸 (arginine, Arg) 以1 : 1之比例組成,而改質後的藍藻蛋白則是以天門冬胺酸 (aspartic acid, Asp) 與精胺酸 (arginine, Arg)賴胺酸(lysine, Lys)比例5:2:3組成。
有鑑於於藍藻蛋白其機械強度不高且在生醫材料上的應用上尚不普遍,本實驗試著以水溶性藍藻蛋白,與具備優良生物相容性的透明質酸進行接枝改質,期待改質後的產物可有效提升機械強度,並有助於細胞的生長。
本實驗透過戊二醛與1-(3-二甲氨基丙基)-3-乙基碳二亞胺作為交聯劑,接枝後的產物可藉由藍藻蛋白上的胺基形成3D立體支架, 並以螢光染色法證實,中國倉鼠卵巢(Chinese hamster ovary, CHO)細胞能貼附在此支架上。
綜合以上,藍藻蛋白接枝透明質酸的立體支架而後將有助於模擬細胞在生物體上生長之情形,甚至可能對細胞生長有益處。
Cyanophycin granule polypeptide (CGP) is a non-ribosomal protein synthesis, first found in 1887 BC when Borzi observed the Cyanobacteria under a microscope.
Because the yield rate of CGP is too low by culturing Cyanobacteria, we implant the cyanophycin synthetase gene of Cyanobacteria into Escherichia coli (E. coli) and use Isopropyl β-D-1-thiogalactopyranoside (IPTG) or Lactose induction to increase the yield rate. Nature CGP consist of aspartic acid (Asp) and arginine (Arg) in ratio 1:1, but recombicant CGP consist of Asp, Arg, and lysine (Lys) in ratio 5:2:3.
Due to its mechanical strength CGP is not popular biomaterials. this experiment use water-soluble CGP to graft hyaluronic acid with excellent biocompatibility ,and expect that modification can effectively enhance both the mechanical strength and the growth of cells.
By glutaraldehyde and 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide(EDC)/N-hydroxysulfosuccinimide(NHS) cross-linking, we use Lys group in CGP to produce HA-CGP 3D scaffold.The 3D scaffold which were observed under a fluorescence microscopy after fluorescence staining were proved that cell could adhesion on this 3D scaffold.
In conclusion, the scaffold could simulate cell growth in vivo, and could help cell growth.
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