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研究生:陳亭羽
研究生(外文):Ting-Yu Chen
論文名稱:利用蛋白質體學方式探討紫雲膏對於傷口癒合之修復過程的影響及功效
論文名稱(外文):The Effect of Shiunko on Wound Healing Using Proteomic Approach
指導教授:翟建富翟建富引用關係
指導教授(外文):Kin-Fu Chak
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
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:96
中文關鍵詞:紫雲膏蛋白質體學傷口癒合
外文關鍵詞:ShiunkoProteomicWound Healing
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紫雲膏 (Shiunko) 為一傳統的植物性中草藥軟膏,在中國和日本已廣泛使用約有數百年的歷史,其主要成分是由紫草的根部及當歸所製成,而在臨床上紫雲膏通常用於治療皮膚的割傷、擦傷、凍傷、燙傷等。 近年來,也有研究指出紫雲膏不僅能夠緩解發炎、疼痛和殺菌,也能夠促進上皮的再生及傷口的癒合,然而,至目前為止,紫雲膏的研究僅止於動物實驗或臨床上的使用。至今,對於紫雲膏療效的相關分子機制還有待進一步的探討。因此,本研究主要目的為利用蛋白質體學及生化功能性分析的方式探討紫雲膏如何參與在傷口的癒合及其可能的作用機轉。
首先,利用95 %酒精將紫草與當歸依重量為1:1 的比例混合萃取,經由減壓濃縮與冷凍乾燥後,將所得到之粉末狀紫雲膏溶於DMSO中,製備成實驗用紫雲膏,並進行細胞存活測試,以找出紫雲膏促進細胞存活的最佳濃度。接著,利用二維電泳實驗找出加藥後表現量有差異的蛋白質,進行蛋白質身分鑑定和功能歸類分群,結果可發現其細胞中有差異表現之蛋白質大多是與抗氧化、細胞增生、抗細胞凋亡和細胞移動相關的蛋白質。 為了進一步闡明紫雲膏在傷口癒合過程中的影響,根據蛋白質體學分析結果,將纖維母細胞經紫雲膏處理後,測定其細胞中活性氧物質含量、細胞增生情況、傷口癒合及細胞遷移能力、膠原蛋白分泌量,實驗結果指出相對於DMSO組,紫雲膏能夠有效降低細胞中活性氧物質的含量,並顯著促進細胞增生、存活和膠原蛋白的分泌,且細胞中TGF-β表現量也有明顯的增加且具有劑量相關性;在細胞移動方面,加入紫雲膏後,會顯著降低細胞的移動性;而動物實驗更進一步確認紫雲膏確實能夠顯著地縮小傷口,以促進傷口癒合。
綜合以上所有實驗結果,在細胞實驗及動物實驗中,紫雲膏可能是透過增加纖維母細胞的抗氧化能力、促進纖維母細胞的增生,並降低細胞移動能力,使得傷口附近的細胞能夠停留在受傷處,大量分泌膠原蛋白用於填補受傷的傷口,進而促進傷口的癒合。
在本研究中,除了發現在蛋白質表現上紫雲膏的新穎作用外,並且也找出紫雲膏參與在傷口癒合中的可能機制。另一方面,此研究不但提供了蛋白質體學作為中草藥研究分析的平台外,同時也表明了在傳統中草藥研究中,以蛋白質體學為基礎的策略優勢,為傳統中草藥的研究提供了一嶄新的方向。

Shiunko is a traditional Chinese herbal medicine which is commonly used in China and Japan. It was mainly composed of Lithospermi radix (LR) and Angelica sinensis radix (AS), and has been used clinically for hundred years in treating wounded skin caused by cuts, abrasions, frost or burn. The previous studies indicated that Shiunko not only has the effects of reduction of inflammation and pain, but also promotes re-epithelialization and wound healing. Although the pharmacological significance about Shiunko was extensively studied, but the mechanism is remain to be clarify. Therefore, the main purpose of this study is to investigate the mechanism and effects of Shiunko involved in wound healing process with proteomic and biological approaches.
Here, Shiunko extract was prepared with 1:1 weight of LR/AS mixture and extracted by 95 % ethanol, then carried out decompression concentration, freeze drying and finally dissolved in DMSO. Cell viability of Shiunko was tested for safety and efficacy concentrations. After that 2D-PAGE was also carried out and the differential displayed proteins were identified for functional clustering analysis. The results of proteomic analysis indicated that these proteins were dominantly participated in anti-oxidant, cell proliferation, anti-apoptosis and cell motility. In order to elucidate the effect of Shiunko on wound healing process, fibroblasts with Shiunko treatment was evaluated by ROS contents, EdU proliferation, wound healing, migration and collagen secretion assay. These results showed that ROS content in cells was substantially reduced, and cell proliferation rate, viability rate and collagen secretion were significantly increased with Shiunko treatment compared with DMSO. Furthermore, the expression of TGF-β was also dramatically increased in a dose dependent manner. On the contrary, the ability of cell migration was significantly decreased with Shiunko treatment. Moreover, animal experiment further confirmed that Shiunko could accelerate the speed of wound closure to promote wound healing.
Taken together, these results suggested that Shiunko really has an enhancing effect associated with wound healing process on fibroblasts both in vitro and in vivo, such as proliferation, anti-oxidant activity, maintenance of cell migration in the injured tissue, and protmotion of collagen secretion.
This study revealed the novel effects of Shiunko on protein expression of fibroblasts, from which a probable molecular based mechanism involved in wound healing process induced by Shiunko is proposed. Thus, in this work has provided a feasible proteomic platform technology for the analysis of the complicated traditional herbal medicine. In addition, this platform technology further illustrate the advantage of proteomics-based strategy in traditional herbal medicine research.

致謝......................................................i
目錄.....................................................iii
圖表目錄 .................................................v
Abstract .................................................vi
中文摘要................................................. ix
第一章、緒論..............................................1
一、皮膚構造、組成及功能..............................1
二、傷口癒合之過程....................................2
三、纖維母細胞在傷口修復過程中所扮演之角色............4
四、紫雲膏及其重要成分紫草、當歸......................4
五、蛋白質體學於生物醫學上之應用......................9
六、研究動機與目的...................................10
第二章、實驗材料與方法...................................12
一、人類纖維母細胞培養...............................12
二、藥物處理.........................................13
三、高壓液相層析儀(HPLC)分析.........................15
四、細胞存活測試.....................................16
五、二維蛋白質電泳分析...............................18
六、快速銀染法.......................................21
七、蛋白質膠內酵素水解...............................22
八、蛋白質影像軟體分析...............................24
九、細胞內蛋白質萃取及定量...........................25
十、SDS-PAGE 蛋白質電泳分析.........................27
十一、西方墨點法及蛋白質轉漬.........................29
十二、細胞內活性氧物質測定...........................31
十三、細胞增生測試...................................32
十四、細胞傷口癒合能力測定...........................33
十五、細胞遷移實驗...................................34
十六、可溶性膠原蛋白測定.............................36
十七、動物實驗.......................................37
第三章、實驗結果.........................................39
一、HPLC分析之結果.................................39
二、自製紫雲膏對於細胞存活率之影響...................39
三、二維電泳展開之蛋白質圖譜及生物資訊學分析.........40
四、西方墨點法確認二維電泳之結果.....................42
五、細胞內活性氧物質含量分析.........................43
六、細胞增生測試分析.................................44
七、細胞傷口癒合能力分析.............................45
八、細胞遷移能力之分析...............................45
九、TGF-β 蛋白質表現量及膠原蛋白分泌量之分析.........46
十、動物實驗之傷口癒合的效果.........................47
第四章、討論..... .......................................49
一、自製紫雲膏成分分析及對細胞存活率影響之探討.......49
二、以蛋白質體學分析紫雲膏可能之作用機制並探討其功能
A. 與抗氧化相關之蛋白質作用.....................50
B. 與調節細胞存活(抗細胞凋亡、細胞增生)相關之
蛋白質作用...................................52
C. 與細胞移動或膠原蛋白分泌相關之蛋白質作用.....55
D. 與代謝相關之蛋白質作用.......................57
三、各組別藥材療效之綜合比較.........................59
第五章、結論...........................................60
參考文獻.................................................62

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