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系統識別號 U0026-0812200911430856
論文名稱(中文) 探討丹參酚酸B對血管平滑肌細胞的移行與增生能力之影響
論文名稱(英文) The effects of Salvianolic acid B on the migration and proliferation of vascular smooth muscle cells
校院名稱 成功大學
系所名稱(中) 細胞生物及解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 93
學期 2
出版年 94
研究生(中文) 李佳玲
研究生(英文) Chia-Lin Lee
學號 T9691103
學位類別 碩士
語文別 中文
論文頁數 69頁
口試委員 指導教授-江美治
口試委員-陳麗玉
口試委員-陳玉怜
中文關鍵字 丹參  丹參酚酸B  平滑肌細胞移行 
英文關鍵字 migration  salvianolic acid 
學科別分類
中文摘要   動脈粥狀硬化是一個形成原因極為複雜的血管疾病,為冠狀動脈疾病及中風的重要致病機轉。平滑肌細胞由血管中層往內皮下間隙移行與增生是形成動脈硬化斑的重要步驟。目前研究指出,氧化壓力在動脈硬化形成初期亦扮演重要角色。丹參,傳統醫學中屬於「活血化瘀」的藥物,廣泛用於治療心血管類疾病,而丹參酚酸B為丹參根莖萃取且具抗氧化能力的水溶性成分。研究證據指出,丹參酚酸B能抑制低密度脂蛋白受到氧化與防止內皮細胞受損,但丹參酚酸B對血管平滑肌的影響目前則尚未清楚。本實驗擬探討丹參酚酸B對培養的血管平滑肌細胞其增生與移行能力之影響。首先,以濃度達100 mM的丹參酚酸B處理72小時對於大白鼠(RASMC)或人類胸主動脈平滑肌細胞(HASMC)並無細胞毒性。而不論以MTT assay或是計算細胞數目的方法,兩種細胞對於25至100 mM丹參酚酸B的處理,都顯示由血清刺激所產生的細胞增生現象受到抑制。另外在細胞的移行實驗中,25至100 mM丹參酚酸B前處理24小時後,PDGF與IGF引起的細胞移行皆被抑制,而癒傷試驗的結果也顯示50 mM 丹參酚酸B會抑制細胞的移行能力。進一步為探討丹參酚酸B在細胞移行的作用,以附著試驗檢測丹參酚酸B是否藉由對平滑肌附著能力的影響而抑制其移行,結果顯示PDGF與丹參酚酸B對於HASMC在膠原蛋白覆層的附著能力並無顯著的促進或抑制。最後以西方墨點轉漬法偵測丹參酚酸B對於PDGF與IGF引起ERK1/2及p38 MAPK磷酸化的影響。實驗結果指出PDGF的刺激具有引起ERK1/2及p38 MAPK磷酸化的效果且達到顯著差異;而100 mM丹參酚酸B前處理24小時則只抑制了p38 MAPK的磷酸化,ERK1/2的磷酸化程度雖有下降,但並無顯著差異。另外,初步的結果顯示在RASMC,25 mM丹參酚酸B前處理24小時可抑制Ang II刺激細胞超氧化物的生成作用。以上的結果顯示Sal B可抑制血管平滑肌細胞的移行與增生。此作用提供了丹參酚酸B治療動脈粥狀硬化的部分機轉。
英文摘要  Atherosclerosis, a complex and multifactorial disease, is the underlying mechanism for vascular disorders including coronary artery disease and stroke. Vascular smooth muscle cells (VSMC) proliferation and migration from media to subendothelial intima plays a major role in the lesion formation of atherosclerosis. Oxidative stress is considered playing an essential role in the initiation and progression of atherosclerosis. Salvia miltiorrhiza (SM) Bunge is a Chinese herb widely used for the treatment of atherosclerosis-related disorders. Salvianolic acid B (Sal B), a potent water-soluble antioxidant isolated from the roots of SM Bunge was demonstrated to inhibit low-density lipoprotein oxidative modification and prevent endothelial damage. The effects of Sal B on smooth muscle cells remain unclear, however. This study examined the effects of Sal B on migration and proliferation of cultured vascular smooth muscle cells. First, treatment of Sal B up to 100 mM and 72 hours exhibited no toxicity on human aortic smooth muscle cell (HASMC) and rat aortic smooth muscle cell (RASMC). Serum-induced proliferation was inhibited by Sal B (25~100 mM) treatment, which was analyzed by MTT assay and cell counting. Pretreatment of Sal B (25~100 mM) for 24 hours attenuated the platelet-derived growth factor (PDGF 0.3 nM) - and insulin-like growth factor 1 (IGF-1 10nM) -induced migration. In addition, wounding assay showed that 50 mM Sal B inhibited the migration activity of HASMC. To elucidate the effects of Sal B on migration, the adhesion of HASMC was examined by adhesion assay. Neither PDGF nor Sal B modulated HASMC adhesion on collagen-coated plates. Pretreatment of 100 mM Sal B inhibited PDGF-stimulated phosphorylation of p38 mitogen-activated protein kinase. Similar effects were observed with extracellular signal-regulated kinase 1/2 though the difference did not reach statistical significance. Preliminary results showed that pretreatment of 25 mM Sal B diminished superoxide production of RASMC induced by angiotensin II. These results demonstrated that Sal B is effective in inhibiting both the proliferation and migration activity of VSMC. These actions are likely to account for part of mechanisms underlying the therapeutic effects of Sal B to treat atherosclerosis.
論文目次 中文摘要 ----------------------------------------------- I
Abstract ----------------------------------------------- III
致謝....................................................V
目錄 --------------------------------------------------- VI
緒論 --------------------------------------------------- 1
材料與方法 --------------------------------------------- 11
一、 細胞培養 ------------------------------------- 16
二、 免疫螢光染色法 ------------------------------- 20
三、 細胞存活率及毒性分析 ------------------------- 22
四、 平滑肌細胞增殖試驗 --------------------------- 24
五、 平滑肌細胞的移行試驗 ------------------------- 25
六、 平滑肌細胞的癒傷試驗 ------------------------- 27
七、 平滑肌細胞的附著試驗 ------------------------- 28
八、 平滑肌細胞超氧化物 ( O2- )的生成分析 ---------- 30
九、 西方墨點轉漬法 ------------------------------- 31
十、 數據處理與統計分析 --------------------------- 38
實驗結果 ----------------------------------------------- 39
討論 --------------------------------------------------- 47
參考文獻 ----------------------------------------------- 51
圖 ----------------------------------------------------- 57
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