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研究生: 林恬妤
Tian-Yu Lin
論文名稱: 吳郭魚膠原蛋白與虎杖萃取物在發展生醫材料之潛力
Pioneer characterizations of combining tilapia collagen and Polyonum cuspidatum extracts as potential biomedical materials
指導教授: 郭嘉信
Guo,Jia-Hsin
學位類別: 碩士
Master
系所名稱: 農學院 - 食品科學系所
Department of Food Science
畢業學年度: 106
語文別: 中文
論文頁數: 74
中文關鍵詞: 魚膠原蛋白虎杖抗菌抗發炎纖維母細胞生長因子
外文關鍵詞: fish collagen, Polygonum cuspidatum, antibacterial, anti-inflammatory, fibroblast growth factor
DOI URL: http://doi.org/10.6346/THE.NPUST.FS.021.2018.E11
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  • 膠原蛋白是人體結構組織中含量最豐富的結構性蛋白。適用於傷口敷料,促進傷口修復、防止細菌感染以及快速止血的效果,但由於人與禽畜共通疾病的流行,使得魚類成為膠原蛋白較安全的來源。根據文獻指出中草藥虎杖擁有許多治療功效,可減輕傷口感染,促進傷口癒合。本研究目的是萃取吳郭魚魚皮和魚骨中的膠原蛋白,加上中草藥虎杖萃取物,以體外試驗探討其殺菌、抗發炎及促進纖維母細胞(fibroblast)生長激素分泌的功能。結果顯示:利用掃描式電子顯微鏡 (SEM)觀察兩種不同乾燥方法的膠原蛋白膜結構,熱乾燥的膠原蛋白膜結構平滑無孔洞,冷凍乾燥的膠原蛋白結構整齊多孔洞,可得知膠原蛋白在高溫下容易破壞原有的結構,使其變性。 藉由抗菌實驗結果不僅得知以虎杖丙酮萃取物抗菌能力佳,其次為虎杖95%乙醇萃取物,也發現虎杖適合利用極性高的溶劑萃取,其中以丙酮溶劑萃取虎杖有效的抗菌成份效果為最佳。經過細胞存活試驗,發現經過低濃度的虎杖萃取物培養後,可促進纖維母細胞的分裂生長,不刺激巨噬細胞引發發炎反應。更進一步分別進行抗發炎與纖維母細胞生長因子試驗,其結果顯示低濃度虎杖水萃取物能夠有效刺激纖維母細胞生長因子的分泌與減少巨噬細胞生成TNF-α,具有降低發炎的功效。綜觀上述研究,魚膠原蛋白適合採用冷凍乾燥方式製作魚膠原蛋白膜,可保持膠原蛋白結構與性質,降低變性風險。虎杖具有良好的抗菌功效之外,能降低發炎,並有促進纖維母細胞生長因子的生成,未來將可運用於製作照護傷口的敷料上。

    Collagen is the most abundant in the structure of the human body structural proteins. Apply to wound dressings, wound repair, prevention of bacterial infections and rapid hemostatic effect. But because of the common prevalence of human and livestock diseases, making fish become a safe source of collagen. According to the literature, the Chinese herbal medicine Polygonum cuspidatum has many therapeutic effects, which can reduce wound infection and promote wound healing. In current research, extraction of collagen from tilapia’s skin and bone, coupled with Chinese herbal extracts of P. cuspidatum were used for in vitro experiments to explore their sterilization, anti-inflammatory and promote fibroblast growth hormone secretion function. The results show that with a scanning electron microscope (SEM) observed two kinds of collagen film structures of different drying methods, heating-dry collagen film is smooth structure without holes, freeze-dry collagen film is multi-mesh holes, it can be seen that collagen easily degrades the original structure at high temperatures and denatures it. According to the results of the antibacterial experiment, it is not only known that the antibacterial ability of the water extract of P. cuspidatum is good, followed by the 95% ethanol extract of P. cuspidatum, it was also found that the P. cuspidatum was suitable for the use of polar high solvent extraction, in which the effective antibacterial component of the P. cuspidatum was best extracted by acetone solvent. After the cell survival test, it was found that after a low concentration of P. cuspidatum extract cultured, fibroblasts could be promoted to divide and grow, but had no significant effect on the growth of macrophages and doesn’t stimulate macrophages to trigger an inflammatory response. Further anti-inflammatory and fibroblast growth factor tests were performed. The results showed that the low concentration of P. cuspidatum aqueous extract can effectively stimulate the secretion of fibroblast growth factor and effectively reducing macrophage production of TNF-α has the effect of reducing inflammation. Looking at the above research, fish collagen is suitable for the production of fish collagen membrane by freeze-drying method, which can maintain the structure and properties of collagen and reduce the risk of degeneration. In addition to its good antibacterial effect, P. cuspidatum can reduce inflammation and promote the production of fibroblast growth factor, which will be used in the future to make dressings for wound care.

    中文摘要.........I
    Abstract........III
    謝誌.............V
    目錄.............VI
    圖目錄...........IX
    表目錄........... X
    1. 前言.........1
    2. 文獻回顧......2
    2.1 膠原蛋白之簡介......2
    2.1.1 膠原蛋白介紹 2
    2.1.2 膠原蛋白之結構......2
    2.1.3 膠原蛋白來源 3
    2.1.4 膠原蛋白的應用......4
    2.2 傷口敷料之簡介........9
    2.2.1 傷口敷料介紹........9
    2.2.2 不同種類的傷口敷料應用及優缺點......10
    2.3 虎杖之簡介....14
    2.3.1 虎杖介紹....14
    2.3.2 虎杖主要的成分......14
    2.3.3 虎杖的功效與應用.....14
    2.4腫瘤壞死因子-α (TNF-α)之簡介....17
    2.4.1 TNF-α介紹.....17
    2.4.2 TNF-α對傷口癒合的影響........17
    2.5鹼性纖維母細胞生長因子之簡介......19
    2.5.1 FGF-2介紹...19
    2.5.2 FGF-2對傷口癒合的影響........20
    3. 材料與方法.....21
    3.1 本實驗之研究架構圖.....21
    3.2 實驗材料......23
    3.2.1 原料.......23
    3.2.2 實驗試藥....23
    3.2.3 培養基......23
    3.2.4 試驗菌株及來源......23
    3.2.5 細胞用試劑.........24
    3.2.6 抗體試劑...........24
    3.2.7 試驗細胞及來源......24
    3.2.8 實驗儀器....24
    3.3魚膠原蛋白分析..26
    3.3.1 魚膠原蛋白萃取......26
    3.3.2 魚膠原蛋白水分含量測定........26
    3.3.3 魚膠原蛋白蛋白質含量測定.......26
    3.3.3.1 樣品前處理和蛋白質含量測定方法......26
    3.3.3.2 蛋白質含量標準曲線...27
    3.3.4 魚膠原蛋白膜結構觀察....27
    3.4 虎杖對金黃色葡萄球菌抗菌實驗方法....28
    3.4.1 虎杖萃取製備 ........28
    3.4.2 金黃色葡萄球菌培養...28
    3.4.3 虎杖萃取對金黃色葡萄球菌抗菌試驗.......28
    3.5虎杖對巨噬細胞存活試驗和TNF-α ELISA試驗...29
    3.5.1 虎杖萃取物樣品製備.......29
    3.5.2 虎杖萃取物對巨噬細胞存活試驗....29
    3.5.3 虎杖萃取物對巨噬細胞TNF-α ELISA測試....30
    3.5.3.1巨噬細胞上清液製備....30
    3.5.3.2 TNF-α細胞激素的ELISA檢測....30
    3.6虎杖對纖維母細胞存活試驗和FGF-2生長因子ELISA試驗....32
    3.6.1虎杖萃取物樣品製備.....32
    3.6.2虎杖萃取物對纖維母細胞存活試驗........32
    3.6.3虎杖萃取物對纖維母細胞FGF-2生長因子ELISA測試.....33
    3.6.3.1纖維母細胞上清液製備..........33
    3.6.3.2 FGF-2生長因子的ELISA檢測....33
    4. 結果與討論............35
    4.1 魚膠原蛋白的一般分析...35
    4.1.1水分含量測定........35
    4.1.2蛋白質含量測定......35
    4.2不同乾燥方法之魚膠原蛋白膜外觀與內部結構的觀察.............37
    4.3不同萃取來源的虎杖萃取物抗菌能力比較與虎杖成分極性探討.......40
    4.3.1不同溶劑的極性大小對虎杖成分的萃取影響..................40
    4.3.2不同萃取來源的虎杖萃取物抗菌比較 .......................40
    4.3.3移除萃取溶劑的虎杖萃取物抗菌比較........................41
    4.4不同萃取來源的虎杖萃取物對巨噬細胞存活試驗..................44
    4.5不同萃取來源的虎杖萃取物對纖維母細胞存活試驗................48
    4.6不同萃取來源的虎杖萃取物對巨噬細胞分泌 TNF-α 激素的影響......52
    4.7不同萃取來源的虎杖萃取物對纖維母細胞分泌FGF-2的影響..........54
    5. 結論.........56
    6. 參考文獻......57
    作者簡介.........63

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