探討化合物Dynasore在活體ICR小鼠與離體CCD-966SK細胞之創傷癒合作用及促人類皮膚纖維母細胞膠原蛋白生成機制

論文目次： 目錄 I 附錄-圖表目錄 III 中文摘要 IV Abstract VII 縮寫(Abbreviation) X 第一章 緒論 1 第一節 人體皮膚構造 1 第二節 皮膚纖維母細胞(human skin fibroblast) 4 第三節 膠原蛋白(collagen) 4 第四節 表皮創傷癒合(Cutaneous wound healing) 6 第五節 Dynasore簡介 9 第六節 MAPK/ERK路徑(Mitogen-activated protein kinase pathway) 10 第七節 MMP-1 (Matrix metalloproteins，MMPs)基質金屬蛋白酵素 13 第八節 TGF-β/SMAD路徑(TGF-β/SMAD pathway) 15 第九節 PI3K/Akt/mTOR路徑(PI3K/Akt/mTOR pathway) 17 第十節研究動機 19 第二章 實驗材料與方法 21 第一節 實驗材料 21 第二節 實驗方法 26 2-1. 活體動物創傷癒合試驗(Wound healing in vivo assay) 30 2-2. 人類皮膚纖維母細胞(Human skin fibroblast)細胞株培養 26 2-3. 人類皮膚纖維母細胞(Human skin fibroblast)細胞株繼代培養 26 2-4. 細胞存活率與生長測定(MTT assay) 27 2-5. 離體創傷癒合試驗(Wound healing in vitro assay) 28 2-6. 細胞內蛋白質製備(Preparation of cellularprotein extracts) 28 2-7. 西方墨點法(Western blotting) 29 2-8. 統計分析(Statistical analysis) 32 第三章 實驗結果 33 第一節 Dynasore對人類皮膚纖維母細胞的存活率與增生實驗 33 第二節 探討Dynasore誘發人類皮膚纖維母細胞磷酸化ERK1/2蛋白質的表現 34 第三節 探討Dynasore誘發受傷人類皮膚纖維母細胞磷酸化ERK1/2蛋白質的表現 35 第四節 探討Dynasore誘發受傷人類皮膚纖維母細胞遷移能力 36 第五節 探討Dynasore誘發正常/受傷人類皮膚纖維母細胞MMP-1蛋白質的表現 37 第六節 探討Dynasore誘發正常/受傷人類皮膚纖維母細胞Procollagen type I蛋白質的表現 39 第七節 探討Dynasore誘發正常/受傷人類皮膚纖維母細胞磷酸化Smad3蛋白質的表現 41 第八節 探討Dynasore誘發正常/受傷人類皮膚纖維母細胞磷酸化Akt蛋白質的表現 42 第九節 探討Dynasore誘發正常/受傷人類皮膚纖維母細胞磷酸化mTOR蛋白質的表現 44 第十節 探討特定抑制劑對Dynasore誘發正常/受傷人類皮膚纖維母細胞蛋白質的表現 46 第十一節 探討Dynasore對於活體動物創傷癒合之影響速率 47 第四章 討論 50 第五章 結論 61 附錄 63 參考文獻 88 參考文獻： Aarbiou, J., Verhoosel, R.M., Van Wetering, S., De Boer, W.I., Van Krieken, J.H., Litvinov, S.V., Rabe, K.F., and Hiemstra, P.S. 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J Biol Chem 278, 51267-51276. ; 膠原蛋白是人體中非常重要的一種蛋白質，存在於結締組織中，具有高張力與拉力強度，是構成真皮、韌帶、肌腱、骨骼與軟骨的蛋白質之一，尤其在人體的皮膚中，是細胞外基質的主要成分。膠原蛋白維持皮膚與肌肉的彈性，且在創傷癒合的過程中扮演著一個促進傷口癒合與組織修護的角色。皮膚創傷癒合主要可以分為發炎反應期、組織增生期及組織重建期。其中，以組織增生期，亦即纖維母細胞增生與膠原蛋白堆積為一關鍵步驟。 Dynasore是一個有機小分子，相關研究文獻仍不多，目前最被確認的角色是，在細胞胞吞作用中，GTP分解酵素，dynamin的抑制劑，Dynasore可直接穿透細胞膜，對於dynamin有較好的專一性。另外，有文獻指出，Dynasore在人類肋膜間皮細胞株中誘發plasminogen activator inhibitor(PAI-1)表現，是經由增強TGF-β訊息傳遞的機制；而TGF-β路徑的訊號分子，除了Smad之外也包含了如MAPK及PI3K路徑等。研究證實，第一型膠原蛋白的形成與TGF-β、PI3K等路徑有關，而Dynasore尚無類似的研究。在本文中，將分別透過離體細胞實驗及活體動物實驗，探討Dynasore在皮膚創傷癒合過程中所造成之影響。 我們發現Dynasore在人類皮膚纖維母細胞株(CCD-966SK)中，即使濃度大於100μM的情況下，亦不具有細胞細胞毒性，並且能誘發人類皮膚纖維母細胞的增生及細胞遷移，此生成作用與Dynasore誘導extracellular signal-regulated kinase (ERK1/2)蛋白質磷酸化及matrix metalloproteinase-1(MMP-1)蛋白質表現有關。此外，我們亦發現，Dynasore選擇性的在創傷細胞中，具有濃度相關性的增加procollagen type I蛋白質的生成。近年來TGF-β/Smad3路徑被認為是調控創傷癒合的路徑之一，且PI3K路徑也與產生collagen type I相關，在Dynasore的作用下，TGF-β/Smad3路徑中的Smad3分子與PI3K中Akt及mTOR 蛋白質的磷酸化，和procollagen type I一樣會在人類皮膚纖維母細胞中被誘發出來。當分別投予ERK、PI3K與mTOR抑制劑, PD98059、LY294002與Repamycin於Dynasore處理之CCD-966SK時，發現只有LY294002能抑制經由Dynasore所誘導增加的Smad3、Akt、mTOR之磷酸化與終產物procolagen type I的表現量，顯示Dynasore所誘發的膠原蛋白之生成是來自活化PI3K/Akt/mTOR pathway。另外，雖然我們也發現PD98059可抑制ERK之磷酸化與MMP-1蛋白之表現量，但是無法抑制procollagen type I之最終生成，因此，我們認為Dynasore雖可正向調控ERK與MMP-1，但此作用可能與膠原蛋白之生成無關。 最後我們利用兩種不同型態的活體動物實驗，來確定Dynasore是否在活體動物之皮膚與黏膜組織上具有促進傷口癒合之功效。結果發現，在皮膚創傷試驗中，與正對照組bFGF相較之下，3 mg/mL的Dynasore於第五天之後其加速小鼠傷口癒合的能力優於bFGF，顯示Dynasore的確有效的促進創傷癒合，而且於某時間點其效用與bFGF相當。至於促進口腔黏膜炎修復，0.3 mg/mL的Dynasore具有一個較控制組更好的癒合效果。 Basic fibroblast growth factor(bFGF)調節多種細胞的生長、分化、遷移和凋亡。在創傷修復中，通過促進與創傷修復有關的幾乎所有細胞的迅速增殖，從而推動創面主動修復，顯著縮短傷口癒合時間，全面提高創傷癒合質量。在本研究中我們所發現的Dynasour不但具有與bFGF相等之作用，更是屬於一個穩定易保存的非蛋白質的化合物。這樣的特質在穩定度與使用保存都較bFGF簡單且方便。 我們結論，在研發創傷癒合藥物時，以Dynasore所具有的高劑量無毒、良好功效與易保存之化合物型態等眾多特質，可能是一個具潛力之候選藥物，我們也嘗試著朝向將其取代bFGF在未來創傷癒合治療中所扮演的角色，甚至應用於癌症化療後之黏膜發炎作用，希望能在此類藥物之研發提供一些新的貢獻。 Collagen is the most abundant protein in humans, it exists in the connective tissue, and with high tension and tensile strength.The collagen molecules assemble into functional integrity of tissue such as skin, ligament, tendon, bone and cartilage which is the main component of the extracellular matrix especially in human skin. Collagen not only maintains the elasticity of skin and muscle but also plays a promoting role during the process of wound healing. Tissue repair has been divided into two phases, one is inflammatory phase, a proliferation phase with synthesis of new connective tissue and epithelial wound closure, the other is maturation phase once the epidermal barrier has been re-established. The most important step of wounding healing is the predominant form of collagen in the human skin and is produced mainly by dermal fibroblasts. Dynasore, a small organic molecule is a newly discovered cell-permeable dynamin inhibitor, blocks the formation of clathrin-coated vesicles through its inhibitory effects on the GTPase activity of dynamin. Studies suggested that Dynasore enhances TGF-β1–induced plasminogen activator inhibitor-1 expression, and might be through JNK signal pathway in human MeT-5A cells. More and more increasing evidences support not only the Smad pathway but also others such as mitogen-activated MAPK and PI3K pathways play the important role in TGF-β signaling. In addition, type I collagen produce relating to TGF-β, PI3K pathway has reperted. In this study, we investigated the effects of Dynasore on human skin fibroblast of wound healing in vitro and in vivo. Results of MTT assay showed that no adverse effect of Dynasore at the concentration up to 100 μM when treated fibroblast. We found Dynasore concentration-dependent increased procollagen type I induction cell proliferation and migration. These effect might be due to induce ERK1/2 and MMP-1 phosphorylation. Recently, TGF-β/Smad3 pathway is recognized in controlling the wound healing, and PI3K also related. Our data suggested Dynasore induced Smad3, Akt, mTOR phosphorylation as well as procollagen type I expression. When treated with ERK, Akt and mTOR inhibitors PD98059, LY294002 and rapamycin, respectively. We found that only LY294002 can inhibit Dynasore -induced procollagen type I protein expression. We conclued that Dynasore -induced procollagen type I protein expression might be through PI3K/Akt pathway. Although PD98059 inhibited the ERK1/2 phosphorylation and MMP-1 expression, but it cannot suppress procollagen type I. indicated that the formation of procollagen type I is indepented to ERK/MAPK pathway and MMP-1. In the in vivo study, ICR mice were created a 6 mm diameter wound on the back by skin biopsy punch. Topical treating with Dynasore, bFGF(basic fibroblast growth factor) as positive control for 14 days, then recorded the wound area every day. Our results showed 3 mg/mL Dynasore significantly promoted wound healing better than bFGF after Day 5. In the mucositis animal model, 0.3 mg/mL Dynasore shown a better outcome than control group. Basic fibroblast growth factor(bFGF) regulates proliferation, differentiation, migration and apoptosis. BFGF has the potential to accelerate wound healing and improve the quality of wound healing by regulating the balance of ECM synthesis and degradation. In this study, Dynasore has the equivalent role of bFGF, is also a non-protein compound, and stable when storage.We suggest that the effectiveness of Dynasore including non-toxic in high dose, excellext effect and easy to preserve. It might be a potential drug candidant in the future. The purpose of this study is to find out a non-protein compound to replace the use of bFGF on wound healing and anti-mucositis, we also wish can develop a good and potential chemical for medical purpose in the drug development.