血管異常性重塑，例如動脈粥狀硬化或血管內膜異常增生造成之血管狹窄，為導致梗塞性腦中風、心肌梗塞或自體動靜脈廔管失敗之主要原因。其中，血管平滑肌細胞的型態轉換在疾病進程扮演關鍵作用。在先前研究中，正丁烯基苯酞在粥狀動脈硬化細胞及動物模式中被證實可抑制血管平滑肌細胞的增殖並促進其細胞凋亡。本研究進一步探討血小板衍生生長因子誘導血管平滑肌細胞模式，及在自體動靜脈廔管動物模式中探討正丁烯基苯酞對血管平滑肌細胞型態之影響。於細胞模式中，本研究觀察到正丁烯基苯酞可抑制血小板衍生生長因子誘導的血管平滑肌細胞增殖，並使其停滯在G0/G1期。此外，亦發現正丁烯基苯酞可抑制血小板衍生生長因子引發之血管平滑肌細胞骨架之重組、增強平滑肌肌動蛋白及膠原蛋白表現量，並抑制血小板衍生生長因子誘導加強的細胞遷移能力。於大鼠自體動靜脈廔管模式中，血栓、血管狹窄、以及增強的血管平滑肌細胞肌動蛋白與膠原蛋白表現量皆受到正丁烯基苯酞的抑制，表示正丁烯基苯酞可抑制血小板衍生生長因子誘導之型態轉換及遷移。此外，研究發現正丁烯基苯酞對平滑肌細胞型態轉換的抑制作用來自於腺苷單磷酸活化蛋白質激酶之活化與哺乳動物雷帕黴素靶蛋白之抑制。藉由基因沉默方式抵消正丁烯基苯酞對腺苷單磷酸活化蛋白質激酶的作用後，發現血小板衍生生長因子誘導血管平滑肌細胞之型態轉換現象被加劇，證實了正丁烯基苯酞可透過腺苷單磷酸活化蛋白質激酶訊息傳遞路徑調控血管平滑肌細胞型態轉換，有助於成為具血管保護潛力之新藥。

The aberrant vascular remodeling, such as atherosclerosis or abnormal neointimal hyperplasia, is the main cause of ischemic stroke, myocardial infarction or failure of arteriovenous fistula. The phenotypic switch of vascular smooth muscle cells (VSMCs) plays a pivotal role in the pathological development. In our previous study, n-butylidenephthalide (BP) was reported to have anti-proliferating and apoptotic effects on VSMCs. The purpose of the current study is to further investigate its role in platelet-derived growth factor (PDGF-bb)-induced VSMC phenotypic modulation in an arteriovenous fistula model. In vitro, we observed that BP inhibited the PDGF-bb-induced cytoskeleton reorganization of the VSMCs. The enhanced expression of vimentin and collagen I, as well as the migration ability induced by PDGF-bb, were also inhibited by BP. By cell cycle analysis, we found that BP inhibited the PDGF-bb-induced VSMCs proliferation and arrested the VSMCs in the G0/G1 phase. In an arteriovenous fistula rat model, the formation of stenosis, which was coupled with a thrombus, and the expression of vimentin and collagen I in VSMCs, were also inhibited by administration of BP, indicating that BP inhibited the PDGF-bb-induced phenotypic switch and the migration of VSMCs. Besides, the inhibitory effects of BP on the phenotypic switch were found to accompany the activated 5’ AMP-activated protein kinase (AMPK) as well as the inhibited phosphorylation of mTOR. Knockdown of AMPK by gene silencing conflicted the effects of BP and further exacerbated the PDGF-bb-induced VSMCs phenotypic switch, confirming the modulating effect that BP exerted on the VSMCs by this pathway. These findings suggest that BP may contribute to the vasculoprotective potential in vasculature.

一、中文摘要 1
二、英文摘要 3
三、前言 7
1. 末期腎臟病與血液透析治療概況 7
2. 自體動靜脈廔管 7
3. 自體動靜脈廔管內層異常增生及狹窄之主要致病機制 8
4. 血管平滑肌細胞為血管內層狹窄的主要作用細胞 10
5. AMP-activated protein kinase (AMPK) 12
6. 正丁基烯基苯酞 (Butylidenephthalide, BP) 13
四、研究目的 14
五、材料與方法 15
1. 細胞培養與繼代 15
2. 血管平滑肌細胞型態轉換誘導 16
3. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 分析法 16
4. BP抑制血管平滑肌細胞遷移能力之分析 17
5. 細胞週期分析 18
6. 免疫螢光染色 18
7. 全細胞總蛋白質抽取及蛋白質濃度定量 19
8. 蛋白質電泳及西方墨點轉印分析 20
9. 小分子干擾核糖核酸(small interfering RNA，siRNA)設計及細胞轉染 22
10. 自體動靜脈廔管動物模式 23
11. 組織處理及切片 24
12. 常規(H&E stain)及免疫組織化學(IHC stain)染色分析 25
13. 統計分析 26
六、結果 27
1. BP抑制PDGF-bb誘導血管平滑肌細胞A7r5之增殖 27
2. BP抑制 PDGF-bb誘導血管平滑肌細胞A7r5型態之轉換 28
3. BP抑制PDGF-bb誘導血管平滑肌細胞之遷移 29
4. BP抑制大鼠自體動靜脈廔管血栓形成及內層狹窄 29
5. BP抑制大鼠自體動靜脈廔管靜脈端血管平滑肌細胞型態之轉換 30
6. BP透過AMPK-mTOR路徑抑制血管平滑肌細胞A7r5型態轉換 31
七、討論 33
八、結論 38
九、結果圖與表 39
十、補充資訊 52
十一、參考文獻 54

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