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研究生:葉重如
研究生(外文):Chung-Ju Yeh
論文名稱:Artocarpin促進皮膚傷口癒合
論文名稱(外文):Artocarpin Enhances Skin Wound Healing
指導教授:王淑慧王淑慧引用關係
指導教授(外文):Shu-Huei Wang
口試委員:龔秀妮王仰高陳金銓
口試委員(外文):Siu-Ni Kung
口試日期:2015-07-03
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:解剖學暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:54
中文關鍵詞:傷口癒合黃酮類化合物纖維母細胞角質細胞內皮細胞膠原纖維細胞增生細胞移動血管新生
外文關鍵詞:artocarpinNIH3T3HaCaTHUVECcollagencell proliferationcell migrationangiogenesis
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皮膚可以幫助身體抵抗外來的有害物質和微生物。當皮膚受到傷害時,傷口癒合的過程需要被精密調控以恢復皮膚組織的正常功能。Artocarpin,一種從植物Artocarpus communis中萃取出來的異戊二烯化之黃酮類化合物(flavonoid),在先前的研究中發現有抗發炎和抗腫瘤的效果。本次實驗的目標是要探討artocarpin在傷口癒合中可能的療效和相關的作用機制。

本次實驗中,artocarpin在小鼠的切除傷口模式中加速了傷口癒合。嗜中性球和巨噬細胞的免疫組織化學染色以及小鼠的cytokine array顯示artocarpin經由加速傷口癒合中發炎期的過程來達到調控發炎的效果。在Mallory苯胺藍膠原蛋白染色、天狼星紅苦味酸染色和西方墨點法的實驗結果中,在以artocarpin治療的組別,膠原纖維的堆積有增加的情形。此外,在cytokeratin14 (CK14,角質細胞marker)和proliferating cell nuclear antigen (PCNA)的雙重染色結果顯示下,artocarpin可以經由促進角質細胞的增生來促進表皮再生的過程。在CD31(血管內皮細胞marker)的免疫組織化學染色和西方墨點法實驗結果顯示,以artocarpin治療的組別,有血管新生增加的情形。在細胞實驗中,纖維母細胞、角質細胞和血管內皮細胞在以artocarpin處理後,細胞增生都有顯著的增加,而這些作用可能是藉由extracellular signal–regulated kinase 1/2 (ERK1/2)、P38或Akt所進行調控。在scratch wound assay中,以artocarpin處理纖維母細胞和角質細胞加速了其傷口閉合,以artocarpin處理之角質細胞也展現了增強的細胞移動能力。此外,以artocarpin處理血管內皮細胞可以增進其在matrigel tube formation assay中形成脈管構造的能力。

綜合以上結果,我們的研究顯示artocarpin可以促進傷口癒合,此效果可能是經由加速發炎期的過程的進行、促進膠原纖維堆積、表皮再生和血管新生等過程所造成。這些研究結果顯示了在傷口癒合治療上,artocarpin極具有治療的潛力。


Skin protects the body against harmful substances and microorganisms. When skin is damaged, wound healing needs to be finely regulated to restore the normal function of skin tissue. Artocarpin, a prenylated flavonoid purified from the plant Artocarpus communis, has been reported to have anti-inflammation and anti-cancer properties. The aim of the present study is to evaluate the wound healing potential and the relative therapeutic mechanism of artocarpin.

In this study, artocarpin accelerated the wound healing process in a mouse skin excisional wound model. Immunohistochemical staining of neutrophil and macrophage and mouse cytokine array analysis revealed that artocarpin exerts an inflammatory regulation by accelerating the process of inflammatory phase of wound healing. Mallory’s aniline blue collagen stain, picrosirius red stain, and western blot analysis showed increased collagen deposition in the artocarpin-treated group. Furthermore, the results of double staining of cytokeratin 14 (CK-14, a keratinocyte marker) and proliferating cell nuclear antigen (PCNA) showed that artocarpin promote re-epithelialization by enhancing keratinocyte proliferation. Immunohistochemical staining and western blot analysis of CD31 revealed an increase in angiogenesis in the artocarpin-treated group. In the in vitro study, increased proliferation of fibroblasts, keratinocytes, and endothelial cells were observed when treated with artocarpin, and these effects were mediated through activation of extracellular signal–regulated kinase 1/2 (ERK1/2), P38, or Akt. Keratinocytes and fibroblasts treated with artocarpin showed accelerated wound closure in scratch wound assay. Keratinocytes treated with artocarpin also exhibited increased cell migration. Furthermore, artocarpin treatment enhanced the ability of endothelial cells to form tubes in matrigel tube formation assay.

Taken together, our studies suggest that artocarpin enhances skin wound healing, possibly through accelerating the inflammatory phase, as well as increasing collagen deposition, re-epithelialization, and angiogenesis. These studies implicate artocarpin as a potential powerful therapeutic agent for the treatment of skin wounds.


口試委員審定書…………………………………………………………………I
致謝………………………………………………………………………………II
目錄………………………………………………………………………………III
圖目錄……………………………………………………………………………V
中文摘要…………………………………………………………………………VI
英文摘要………………………………………………………………………VII
壹、 緒論………………………………………………………………………1
一、 引言………………………………………………………………1
二、 皮膚傷口癒合的機制……………………………………………1
三、 發炎期……………………………………………………………1
四、 增生期……………………………………………………………2
五、 重塑期……………………………………………………………2
六、 細胞增生的分子路徑……………………………………………2
七、 Artocarpin………………………………………………………3
貳、 實驗材料…………………………………………………………………4
參、 實驗方法………………………………………………………………10
一、 傷口模式…………………………………………………………10
二、 細胞培養…………………………………………………………10
三、 石蠟切片包埋……………………………………………………10
四、 冷凍切片包埋……………………………………………………11
五、 蘇木紫-伊紅染色(Hematoxylin& Eosin stain) ………………11
六、 免疫組織化學染色(Immunohistochemistry) …………………12
七、 Mouse cytokine array…………………………………………12
八、 Mallory苯胺藍膠原蛋白染色
(Mallory’s aniline blue collagen stain) ………………………13
九、 天狼星紅苦味酸染色(Picrosirius red stain) …………………13
十、 西方墨點法(Western blot) ……………………………………13
十一、 流式細胞儀分析(Flow cytometry analysis) …………………14
十二、 結晶紫染色(Crystal violet stain) ………………………………14
十三、 MTT assay………………………………………………………15
十四、 Scratch wound assay……………………………………………15
十五、 BrdU免疫細胞化學染色………………………………………15
十六、 Matrigel tube formation assay…………………………………16
十七、 統計分析…………………………………………………………16
肆、 實驗結果…………………………………………………………………17
一、 Artocarpin具有加速皮膚傷口癒合的效果 …………………17
二、 Artocarpin具有加速發炎細胞進入傷口與清除的效果………17
三、 Artocarpin對發炎期中cytokine的表現影響…………………17
四、 Artocarpin具有促進膠纖維堆積的效果………………………18
五、 Artocarpin藉由活化Akt和ERK分子路徑促進纖維母細胞增生……………………………………………………………………18
六、 Artocarpin不影響纖維母細胞的移動…………………………19
七、 Artocarpin藉由活化ERK分子路徑促進角質細胞增生……19
八、 Artocarpin藉由活化ERK分子路徑促進角質細胞移動……20
九、 Artocarpin具有促進血管新生的效果…………………………20
十、 Artocarpin藉由活化ERK和P38分子路徑促進血管內皮細胞增生……………………………………………………………………21
十一、 Artocarpin藉由活化P38分子路徑促進血管內皮細胞形成脈管構造……………………………………………………………………21
伍、 討論………………………………………………………………………22
陸、 附圖………………………………………………………………………26
柒、 參考文獻…………………………………………………………………51


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