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研究生:曾皇諭
研究生(外文):Huang-Yu Zeng
論文名稱:褐藻醣膠修飾幾丁聚醣/褐藻酸鈉支架控制釋放鹼性纖維母細胞生長因子在傷口修復之應用
論文名稱(外文):Fucoidan-modified Chitosan/Alginate Scaffold for Controlled Release of Basic Fibroblast Growth Factor in Wound Healing
指導教授:黃意真黃意真引用關係
指導教授(外文):Yi-Cheng Huang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:100
中文關鍵詞:傷口修復硫酸化褐藻醣膠鹼性纖維母細胞生長因子控制釋放
外文關鍵詞:Wound repairSulfated fucoidanBasic fibroblast growth factorControlled release
相關次數:
  • 被引用被引用:2
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  • 下載下載:101
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燙傷、割傷、擦傷、潰瘍及多種原因會造成皮膚損傷。傷口暴露於空氣中太久,容易遭受微生物感染,因此,近年來研究著重於加速傷口修復。目前已知,鹼性纖維母細胞生長因子 (Basic fibroblast growth factor, bFGF) 可促進纖維母細胞遷移、增生及角質細胞生長,有效的幫助組織的修復、皮膚傷口癒合。但 bFGF 半衰期短且易受酵素降解,因而影響傷口修復之成效。本研究以褐藻醣膠修飾幾丁聚醣/褐藻酸鈉支架保護及控制釋放 bFGF,探討對於傷口修復影響。結果指出:幾丁聚醣及褐藻酸鈉體積比 3:1 製備之支架,具有多孔性及良好膨潤吸水特性,降解速率也較緩慢;將褐藻醣膠 C-4 上的 –OH 基硫酸化後,硫酸化程度提升至 43% (F43),可維持原有之抗氧化特性、有效增加生物相容性,並保護 bFGF 使其免受酵素降解;硫酸化之褐藻醣膠修飾幾丁聚醣/褐藻酸鈉支架具有高度生物相容性、抗發炎特性、攜帶 bFGF 後並延緩其釋放,在 72 小時累積釋放濃度為 2.37 ng/mL,達到維持活性及減低副作用功效,並促進纖維母細胞遷移、增生及角質細胞生長。綜合上述,硫酸化褐藻醣膠修飾幾丁聚醣/褐藻酸鈉支架能有效幫助傷口修復,未來在臨床的應用將極具潛力。
There are many reasons for skin damage like burns, cuts, abrasions and ulcers. Wounds were vulnerable infected by microorganisms if it is exposed to air for a long time. In the recent, the studies focus on accelerating wound repair. It is known that basic fibroblast growth factor (bFGF) promoted fibroblasts migration, proliferation, and keratinocytes growth and effective helped tissue repair and skin wound healing. However, the use of bFGF is limited by two drawbacks, which are it’s short half-life and easily degradated by enzyme. In this study, we fabricate fucoidan-modified chitosan/alginate scaffold to protect and control release bFGF and evaluate effect on wound healing.
The experimental results show that the volume ratio of chitosan to alginate at 3:1 was porious, swellable and degraded slowly. After sulfation of fucoidan, the –OH group on C-4 was replaced by –SO3 and the sulfated degree increased to 43% which is called F43. 1% F43 had effective anti-oxidation properties, good biocompatibility and protected bFGF from trypsin degradation. 1% F43-modified chitosan/alginate scaffold (1% F43-S) are suitable biomaterials because of suitable degradation rate, good biocompatibility, and anti-inflammation properties. Moreover, bFGF loaded 1% F43-S could maintain bFGF activity and control its release. The concentration of bFGF reached 2.37 ng/mL for 72 hours release. In vitro studies show that bFGF loaded 1% F43-S promoted fibroblasts proliferation, migration, and keratinocytes growth. In brief, bFGF loaded 1% F43-S effectively helped the wound repair and will have a great potential in clinical applications in the future.
目錄
謝誌 III
中文摘要 IV
ABSTRACT V
目錄 VI
第一章、 前言 1
第二章、文獻回顧 3
2.1、皮膚組織組成及功能 3
2.2、皮膚傷口分類及修復 4
2.2-1發炎期 5
2.2-2 增生期 6
2.2-3 修復期 7
2.3、生長因子與傷口修復 7
2.3-1 生長因子在傷口修復中之角色 7
2.3-2 鹼性纖維母細胞生長因子功能、應用及限制 8
2.4、組織工程 9
2.4-1 控制釋放 10
2.4-2 支架 11
2.4-3 幾丁聚醣結構及特性 11
2.4-4 褐藻酸鈉結構及特性 12
2.4-5 幾丁聚醣/褐藻酸鈉於生醫材料上應用 13
2.4-6 褐藻醣膠結構、特性及硫酸根特性 13
第三章、實驗目的與動機 15
第四章、實驗設計 16
第五章、材料方法 17
5.1實驗藥品 17
5.2 實驗儀器 19
5.3幾丁聚醣/褐藻酸鈉支架之材料特性試驗 20
5.3-1 幾丁聚醣特性分析 20
5.3-2幾丁聚醣/褐藻酸鈉支架製備 21
5.3-3幾丁聚醣/褐藻酸鈉支架物化特性分析 21
5.4褐藻醣膠修飾幾丁聚醣/褐藻酸鈉支架之材料特性試驗 23
5.4-1不同硫酸化程度褐藻醣膠製備及硫酸根分析定量 23
5.4-2褐藻醣膠清除 DPPH 自由基試驗 24
5.4-3褐藻醣膠對於 bFGF 酵素保護試驗 24
5.4-4褐藻醣膠修飾幾丁聚醣/褐藻酸鈉支架製備及降解試驗 25
5.4-5褐藻醣膠修飾支架控制釋放 bFGF 試驗 25
5.4-6 Enzyme-linked immunosorbent assay (ELISA) 分析步驟 26
5.5褐藻醣膠修飾幾丁聚醣/褐藻酸鈉支架之細胞試驗 27
5.5.1傷口修復發炎期 27
5.5.1-1細胞培養 27
5.5.1-2褐藻醣膠修飾支架之 RAW264.7 細胞存活率試驗 28
5.5.1-3褐藻醣膠之 RAW264.7 細胞存活率試驗 29
5.5.1-4褐藻醣膠修飾支架之抗發炎試驗 29
5.5.2傷口修復增生期 30
5.5.2-1細胞培養 30
5.5.2-2褐藻醣膠修飾支架攜帶 bFGF 對 L-929 細胞存活率之影響 30
5.5.2-3褐藻醣膠對 L-929 細胞存活率之影響 31
5.5.2-4不同 bFGF 濃度對 L-929 細胞存活率之影響 31
5.5.2-5材料對於 L-929 之細胞形態染色實驗 32
5.5.2-6褐藻醣膠修飾支架攜帶 bFGF 對 L-929 細胞遷移之影響 32
5.5.3傷口修復修復期 33
5.5.3-1細胞培養 33
5.5.3-2褐藻醣膠修飾支架攜帶 bFGF 對 HaCaT 細胞存活率之影響 33
5.5.3-3褐藻醣膠對 HaCaT 細胞存活率之影響 34
5.5.3-4不同 bFGF 濃度對 HaCaT 細胞存活率之影響 35
5.5.3-5材料對於 HaCaT 之細胞形態染色實驗 35
第六章、結果與討論 36
6.1幾丁聚醣/褐藻酸鈉支架之材料特性試驗 36
6.1-1幾丁聚醣特性分析 36
6.1-2幾丁聚醣/褐藻酸鈉支架物化特性分析 36
6.2褐藻醣膠修飾幾丁聚醣/褐藻酸鈉支架材料特性試驗 38
6.2-1不同硫酸化程度褐藻醣膠之硫酸根含量分析 38
6.2-2褐藻醣膠清除 DPPH 自由基試驗 39
6.2-3褐藻醣膠對於 bFGF 穩定性及酵素保護試驗 39
6.2-4褐藻醣膠修飾支架之降解試驗 40
6.2-5褐藻醣膠修飾支架控制釋放 bFGF 試驗 41
6.3褐藻醣膠修飾幾丁聚醣/褐藻酸鈉支架之細胞試驗 41
6.3.1傷口修復發炎期 41
6.3.1-1褐藻醣膠修飾支架之 RAW264.7 細胞存活率試驗 41
6.3.1-3褐藻醣膠修飾支架之抗發炎試驗 42
6.3.2傷口修復增生期 43
6.3.2-1褐藻醣膠修飾支架攜帶 bFGF對 L-929 細胞存活率之影響 43
6.3.2-2材料對於 L-929 之細胞形態染色實驗 44
6.3.2-3褐藻醣膠修飾支架攜帶 bFGF 對 L-929 細胞遷移之影響 44
6.3.3傷口修復修復期 45
6.3.3-1褐藻醣膠修飾支架攜帶 bFGF 對 HaCaT 細胞存活率之影響 45
6.3.3-2材料對於 HaCaT 之細胞形態染色實驗 46
第七章、結論 47
第八章、未來展望 48
第九章、參考文獻 49
圖目錄
圖一、傷口分類。 57
圖二、皮膚組織。 58
圖三、傷口修復過程 (A)發炎期 12-24小時 (B)增生期 1-7天 (C)修復期 3-14天。 59
圖四、組織工程之重要因子。 60
圖五、幾丁聚醣化學結構。 61
圖六、褐藻酸鈉化學結構。 62
圖七、褐藻醣膠化學結構。 63
圖八、FT-IR 分析幾丁聚醣/褐藻酸鈉支架之結構。 64
圖九、C1A1、C1A3 及 C3A1 支架之掃描式電子顯微鏡分析於 100X 下表面及截面結構。 65
圖十、 C1A1、C1A3 及 C3A1 支架在 37℃ 含有 Lysozyme (1000 IU/mL) 下之降解特性。 66
圖十一、 C1A1、C1A3 及 C3A1 支架在37℃之膨潤特性。 67
圖十二、FT-IR 分析褐藻醣膠硫酸化前後結構改變。 68
圖十三、以 DPPH 試驗評估不同硫酸化程度及濃度之褐藻醣膠抗氧化活性。 69
圖十四、褐藻醣膠於 37℃ 維持 bFGF 之穩定性。 70
圖十五、褐藻醣膠經胰蛋白酶作用之保護 bFGF 特性。 71
圖十六、酵素影響不同組別 bFGF 前後之差異性。 72
圖十七、褐藻醣膠修飾 C3A1 支架之降解特性。 73
圖十八、褐藻醣膠修飾 C3A1 支架控制釋放 bFGF 特性。 74
圖十九、褐藻醣膠修飾支架對於 RAW264.7 細胞存活率之影響。 75
圖二十、褐藻醣膠對於 RAW264.7 細胞存活率之影響。 76
圖二十一、以 NO 生成量評估不同硫酸化褐藻醣膠修飾支架對於 RAW264.7 細胞之抗發炎能力影響。 77
圖二十二、褐藻醣膠修飾之支架對於 L-929 細胞存活率之影響。 78
圖二十三、F21、F43 對於 L-929 細胞 24、72 小時細胞存活率之影響。 79
圖二十四、不同濃度 bFGF 對於 L-929 細胞存活率之影響。 80
圖二十五、以 Live & Dead Assay 分析 1% F43-Sb、bFGF 及 Control 對於 L-929 細胞於 72 小時在 200X 之細胞形態圖。 81
圖二十六、L-929 細胞於含 0.5% 血清之 DMEM/F12 中生長曲線。 82
圖二十七、褐藻醣膠修飾之支架於 3 小時中對於 L-929 細胞遷移之影響 (A)細胞遷移圖 (B) 量化圖。 83
圖二十八、褐藻醣膠修飾之支架對於 HaCaT 細胞 72 小時細胞存活率之影響。 84
圖二十九、F21、F43 對於 HaCaT 細胞 72 小時細胞存活率之影響。 85
圖三十、不同濃度 bFGF 對於 HaCaT 細胞 72 小時細胞存活率之影響。 86
圖三十一、以 Live & Dead Assay 分析1% F43-Sb、bFGF 及 Control 對於 HaCaT 細胞於 72 小時在 100X 之細胞形態圖。 87
表目錄
表一、支架孔洞直徑大小分析 88
表二、褐藻醣膠硫酸化程度分析 89


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