臺灣博碩士論文加值系統

本實驗探討兩種真菌來源幾丁質包括SACCHACHITIN與RHIZOCHITIN是否可促進糖尿病慢性傷口的癒合，並評估其成效。實驗分別透過高糖細胞模型以及化學藥劑Streptozotocin(STZ)誘導糖尿病動物模型探討傷口癒合機制。細胞增生實驗中，Hs68纖維母細胞株先以高糖培養液(110 mM glucose)培養24小時，再分別加入不同濃度的SACCHACHITIN與RHIZOCHITIN(100, 250, 500 μg/ml )，於7天後以MTS assay分析其細胞存活率。結果顯示，在高糖環境下，SACCHACHITIN與RHIZOCHITIN皆能提高細胞的存活率。以in vitro scratch assay觀察細胞遷移的情形。結果顯示，在高糖環境下，SACCHACHITIN與RHIZOCHITIN亦能促進細胞移行的能力。動物實驗中，於STZ誘導型糖尿病Wistar Han IGS公鼠背部製作全層皮膚傷口(4 cm2)，分別以SACCHACHITIN與RHIZOCHITIN薄膜處理之，分於3、7、14、21天觀察傷口面積的變化。傷口面積試驗顯示，SACCHACHITIN與RHIZOCHITIN皆能促進糖尿病傷口的癒合。實驗進一步於SACCHACHITIN與RHIZOCHITIN中添加血小板破膜後富有生長因子之GF18，製成新的複合創傷敷料，評估其對於糖尿病導致慢性傷口癒合之成效。傷口面積試驗顯示，SACCHACHITIN結合GF18複合敷料與RHIZOCHITIN結合GF18複合敷料更具促進癒合之效果。將取下傷口樣本以酵素連結免疫法(Enzyme-linked immunosorbent assay, ELISA)分析其Transforming growth factor-beta1(TGF-β1)的分泌量，結果顯示，SACCHACHITIN、 RHIZOCHITIN、GF18及新的複合材料皆能在不同時期增加TGF-β1的分泌，且與傷口面積變化的趨勢相似，顯示敷料可能透過調控傷口部位生長因子的含量，達到促進傷口癒合的效果。

In the present study, SACCHACHITIN and RHIZOCHITIN were used to evaluate the effectiveness in accelerating chronic wound healing, by employing a cell line in vitro model and a chemical-induced diabetic rat in vivo model. For In vitro, Hs68 fibroblast cells were cultured in high-glucose medium (110 mM glucose) for 24 hours and then SACCHACHITIN and RHIZOCHITIN (100, 250, and 500 μg/ml) were supplemented as suspension to the medium. After 7 days, we observed both SACCHACHITIN and RHIZOCHITIN resumed cell viability and cell mobility of Hs68 cell in high-glucose condition. In in vivo model, full-thickness skin wounds (4 cm2) were created on the dorsum of wistar diabetic rats. The cutaneous wounds were treated with membrane of SACCHACHITIN or RHIZOCHITIN, and the area of wounds was analysed at 3, 7, 14, 21 days. The data in vivo indicated both SACCHACHITIN and RHIZOCHITIN induced wound healing, significantly. Furthermore, new wound dressings were designed to combine with GF18, a mixture of human platelet growth factor, with SACCHACHITIN or RHIZOCHITIN, and the effects of the novel wound dressings on diabetic wound healing were evaluated. The data in vivo indicated the addition of GF18 optimized the recovery effect of SACCHACHITIN and RHIZOCHITIN. Transforming growth factor-beta1(TGF-β1) was assayed by ELISA in the samples of wound area. As a result, SACCHACHITIN, RHIZOCHITIN, GF18 and new wound dressings all significantly increased the production of TGF-β1 in different stages of the healing process.

中文摘要 I
英文摘要 II
致謝 III
目錄 IV
圖目次 VI
第一章 緒論 1
第二章 文獻回顧 2
第一節 慢性傷口與糖尿病足部潰瘍 2
第二節 皮膚傷口癒合過程 4
第三節 基質金屬蛋白水解酶 6
第四節 Transforming growth factor-β（TGF-β） 8
第五節 幾丁質與幾丁聚醣 8
第六節 幾丁質、幾丁聚醣於傷口癒合之研究 11
第七節 本實驗室所開發之生物性材料 16
第八節 血小板破膜後富有生長因子GF18 18
第三章 實驗架構 19
第四章 材料及方法 22
第一節 實驗材料 22
第二節 實驗方法 24
第一部分 敷料的製備流程 24
一、 SACCHACHITIN的製備 24
二、 菌種(R.stolonifer F6)培養 25
三、 RHIZOCHITIN的製備 26
四、 複合敷材的製備 26
第二部分 細胞實驗 27
一、 細胞培養 27
二、 細胞培養基配置 27
三、 冷凍細胞之解凍與活化 27
四、 細胞繼代培養 27
五、 細胞冷凍保存 28
六、 細胞存活率與增生測試 28
七、 細胞遷徙分析 29
第三部分 動物實驗 29
一、 化學藥劑誘導糖尿病動物模式 29
二、 大傷口(4 cm2)之動物評估模式 30
三、 傷口組織取樣 30
四、 蛋白質萃取與定量 30
五、 Matrix Metalloproteinase zymography 31
六、 生長因子TGF-β1分析 31
七、 組織病理切片 31
八、 數據分析 32
第五章 結果 33
第一節 不同濃度葡萄糖對Hs68細胞存活率的影響 33
第二節 不同濃度SACCHACHITIN對高糖環境下Hs68細胞存活率的影響 33
第三節 不同濃度 RHIZOCHITIN對高糖環境下Hs68細胞存活率的影響 33
第四節 高糖環境下SACCHACHITIN與RHIZOCHITIN對Hs68細胞移行能力的影響 34
第五節 傷口面積之比較 35
第六節 傷口部位基質金屬蛋白酶(MMPs)活性表現 37
第七節 傷口部位TGF-β1生長因子含量比較 39
第八節 傷口組織病理切片觀察 39
第六章 討論 42
第七章 結論 48
參考文獻 49
附圖表 55

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