血管再阻塞(restenosis)發生的原因主要關鍵在於內膜增生，而內膜增生最主要的關鍵在於血管平滑肌細胞的遷移(migration)及增生(proliferation)是造成血管再狹窄的主要原因。在病理發展的過程中，血管平滑肌細胞會受血小板衍生的生長因子刺激的造成血管內膜增生及遷移促進血管再狹窄的發生。台灣四面環海，是一個具有豐富海洋資源的國家。因此，本研究探討海洋藥物[Dihydroaustrasulfone alcohol（DA）及Deep sea water(DSW)]與血管在阻塞相關之研究。DA是首次從珊瑚中分離出來的天然化合物，最近有一些研究指出其具有抗癌和抗炎活性。而DSW是來自台灣花蓮外海662公尺深層海水含有豐富的鎂元素，具有生物活性的礦物。先前研究指出，氯化鎂體外治療有可能影響血管平滑肌細胞的增殖和遷移的潛力。由實驗結果表明，DA和DSW顯著降低汽球損傷誘導的大鼠頸動脈內膜增生模型及顯著抑制增殖和血管平滑肌細胞的遷移。我們的結果進一步表明，DA抑制血管平滑肌細胞的遷移及增殖主要藉由一些訊息傳遞路徑，例如ERK / MAPK，PI3K / AKT和STAT3訊號表現。研究結果指出，DA和DSW能降低汽球損傷誘導的大鼠頸動脈內膜增生，其效果可以通過抑制血管平滑肌細胞增殖和遷移進行調控。依據實驗結果，DA和DSW具有潛在的應用價值做為抗血管再阻塞之藥劑應用於預防血管再阻塞的發生。

Dihydroaustrasulfone alcohol is a marine-derived molecule that is isolated from marine corals and has exhibited anti-cancer and anti-inflammatory activity; however, neither an anti-restenotic effect nor a related mechanism has been reported. Our study is the first to demonstrate the key role of dihydroaustrasulfone alcohol, along with its mechanism of action, in preventing balloon angioplasty-induced neointimal hyperplasia as well as vascular smooth muscle cell (VSMC) proliferation and migration. The major biological processes in restenosis are proliferation and migration of VSMCs, which we assessed using MTT, a wound healing assay, a transwell assay, gelatinase zymography, and a western blot. The results revealed that dihydroaustrasulfone alcohol reduced neointima formation in the artery of rats and significantly inhibited VSMC proliferation and migration through ERK/MAPK, PI3K/AKT, and STAT3 signaling pathways. In addition, dihydroaustrasulfone alcohol decreased the expression levels of MMP-2 and MMP-9. Thus, dihydroaustrasulfone alcohol may be a useful anti-restenosis agent and regulate VSMCs; it may have a role in the prevention and treatment of restenosis after successful angioplasty. In conclusion, our findings identify dihydroaustrasulfone alcohol as being involved in the progression of balloon angioplasty-induced neointimal hyperplasia and suggest that it may represent an effective molecular therapy to reduce the incidence of restenosis. Surprisingly, marine extracts contribute to our arsenal of established and safe drugs and could represent a novel adjunct pharmacological strategy for the prevention of restenosis.

目錄 (Contents)
目錄.......................................................................................................... II
圖目錄 ( List of Figures) ....................................................................... V表目錄 (Table of contents) ..................................................................VII中文摘要...............................................................................................VIII 英文摘要.................................................................................................. X
Part Ⅰ：Dihydroaustrasulfone Alcohol...............................................1
Abstract.....................................................................................................2
1. Introduction..........................................................................................4
2. Materials and Methods........................................................................6
2.1. Design of Dihydroaustrasulfone Alcohol’s experiment.................6
2.2. Materials............................................................................................7
2.3. Cell Culture and MTT Cell Proliferation Assay............................7
2.4. Flow Cytometry.................................................................................8
2.5. Cell Migration Assay.........................................................................9
2.6. In-Gel Gelatinase Zymography......................................................10
2.7. Western Blot.....................................................................................10
2.8. Rat Carotid Artery Balloon Angioplasty.......................................11
2.9. Histopathological Analysis..............................................................12
2.10. Statistical Analysis.........................................................................14
3. Results and Discussion.......................................................................15
3.1. Dihydroaustrasulfone Alcohol Inhibited Platelet-Derived Growth Factor (PDGF)-BB-Induced Vascular Smooth Muscle Cell (VSMC) Proliferation............................................................................................15
3.2. Dihydroaustrasulfone Alcohol Inhibits PDGF-BB-Induced VSMC Migration....................................................................................16
3.3. Effects of Dihydroaustrasulfone Alcohol on the Proliferative and Migration-Associated Proteins in VSMCs...........................................17
3.4. Dihydroaustrasulfone Alcohol Inhibited Neointimal Hyperplasia ..................................................................................................................20
4. Conclusions.........................................................................................22
5. Reference.............................................................................................23
Part Ⅱ：Deep Sea Water......................................................................39
Abstract...................................................................................................40
1. Introduction........................................................................................42
2. Materials and Methods......................................................................44
2.1. Design of Deep sea water’s experiment.........................................44
2.2. Materials and DSW preparation....................................................45
2.3. Rat carotid artery model of balloon angioplasty..........................45
2.4 Histopathological analysis...............................................................46
2.5. Blood biochemical analysis.............................................................48
2.6. Measurement of arterial magnesium content...............................48
2.7 Cell culture........................................................................................49
2.8 Cell proliferation assay (MTT assay) ............................................49
2.9 Cell migration assay (Transwell assay) .........................................50
2.10 Western blot....................................................................................50
2.11 Gelatinase zymography..................................................................51
2.12 Data analysis...................................................................................52
3. Results and Discussion.......................................................................52
3.1 Changes of blood biochemical parameters and body weight.......52
3.2 Effect of DSW on balloon angioplasty-induced neointimal hyperplasia..............................................................................................53
3.3 Magnesium content of the carotid artery.......................................54
3.4 Effects of DSW on VSMC proliferation and migration ..................................................................................................................55
3.5 Effects of DSW on the proliferative and migration associated proteins of VSMCs.................................................................................56
4. Conclusions.........................................................................................59
5. Reference.............................................................................................60
圖目錄 (List of Figures)
Part Ⅰ：Dihydroaustrasulfone Alcohol
Figure 1. The chemical structure of dihydroaustrasulfone alcohol (DA)…………………………………………………………………….29
Figure 2. Dihydroaustrasulfone alcohol inhibits platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cell (VSMC) proliferation……………………………………………………………..30
Figure 3. Dihydroaustrasulfone alcohol inhibited PDGF-BB-mediated cell migration of VSMCs………………………………………………31
Figure 4. Dihydroaustrasulfone alcohol inhibited PDGF-BB-activated signaling cascades in VSMCs…………………………………………...33
Figure 5. The regulations of Dihydroaustrasulfone alcohol on gelatinase activation………………………………………………………………....34
Figure 6. Dihydroaustrasulfone alcohol prevents balloon angioplasty-
induced neointima formation……………………………….…………...35
Figure 7. Schematic representation of the potential mechanisms of Dihydroaustrasulfone Alcohol in regulating PDGF-BB-induced VSMC proliferation and migration……………………………………………...38
Part Ⅱ：Deep Sea Water…………………………………………….66
Figure 1. Inhibitory effect of DSW on balloon angioplasty-induced neointimal hyperplasia…………………………………………………..66
Figure 2. Differences in magnesium content in the balloon-injured carotid artery…………………………………………………………….68

Figure 3. DSW-induced regulation of VSMC viability. Cell viability was analyzed with the MTT proliferation assay……………………………..69
Figure 4. Effects of DSW on VSMC migration. Cell migration was analyzed with transwell assays………………………………………….70
Figure 5. Molecular regulation by DSW of cell growth and migration of VSMCs………………………………………………………………….71












表目錄 (Table of contents)
Table 1. Blood biochemical parameters of SD rats at 14 days after balloon angioplasty (BA)………………………………………………………73

part I
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PART Ⅱ
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