臺灣博碩士論文加值系統

　　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。

　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.

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

Babior BM (1999) NADPH oxidase: an update. Blood 93: 1464-1476.
Chen KJ and Su JS et al. (1999) Practical application on Blood Satsis Studies (in Chinese). People Health Publisher
Chen YH, Lin SJ, Ku HH, Shiao MS, Lin FY, Chen JW and Chen YL (2001) Salvianolic acid B attenuates VCAM-1 and ICAM-1 expression in TNF-alpha-treated human aortic endothelial cells. Journal of Cellular Biochemistry 82: 512-521.
Chen YL, Yang SP, Shiao MS, Chen JW and Lin SJ (2001) Salvia miltiorrhiza Inhibits Intimal Hyperplasia and Monocyte Chemotactic Protein-1 Expression After Balloon Injury in Cholesterol-Fed Rabbits. Journal of Cellular Biochemistry 83: 484-493.
Griendling KK, Minieri CA, Ollerenshaw JD and Alexander RW (1994) Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circulation Research 74: 1141-1148.
Griendling KK, Sorescu D and Ushio-Fukai M (2000) NAD(P)H oxidase: role in cardiovascular biology and disease. Circulation Research 86: 494-501.
Hayashi K, Saga H, Chimori Y, Kimura K. Yamanaka Y and Sobue K (1998) Differentiated phenotype of smooth muscle cells depends on signaling pathways through insulin-like growth factors and phosphatidylinisitol 3-kinase. Journal of Biological Chemistry 273: 28860-28867.
Hayashi K, Takahashi M, Kimura K. Nishida W, Saga H and Sobue K (1999) Changes in the balance of phosphatidylinisitol 3-kinase/protein kinase B(Akt) and the mitogen-activated protein kinases (ERK/p38MAPK) determine a phenotype of visceral and vascular smooth muscle cells. Journal of Cell Biology 145: 727-740.
Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey E, Weber LA and Gerthoffer WT (1999) A role for p38 (MAPK)/HSP27 pathway in smooth muscle cell migration. Journal of Biological Chemistry 274: 24211-24219.
Hung HH, Chen YL, Lin SJ, Yang SP, Shin CC, Shiao MS and Chang CH (2001) A salvianolic acid B-rich fraction of Salvia miltiorrhiza induces neointimal cell apoptosis in rabbit angioplasty model. Histology & Histopathology 16: 175-183.
Irani K (2000) Oxidant signaling in vascular cell growth, death and survival : a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circulation Research 87: 179-183.
Jones RD, Hancock JT and Morice AH (2000) NADPH oxidase: auniversal oxygen sensor? Free Radical Biology & Medicine 29: 416-424.
Jones SA, O'Donnell VB, Wood JD, Broughton JP, Hughes EJ, and Jones OT (1996) Expression of phagocyte NADPH oxidase components in human endothelial cells. American Journal of Physiology 271: H1626-H1634.
Kumar A, Middleton A, Chambers TC and Mehta KD (1998) Differential riles of extracellular signal-regulated kinase-1/2 and p38(MAPK) in interlrukin-1 beta- and tumor necrosis factor-alpha-induced low dendity lipoprotein receptor expression in HepG2 cells. Jounal of Biological Chemistry 273: 15742-15748.
Kyriakis JM and Avruch J (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiological Reviews 81: 807-869.
Lewis TS, Shapiro PS and Ahn NG (1998) Signal transduction through MAP kinase cascade. Advances in Cancer Research 74: 49-139.
Nishio E and Watanabe Y (1997) The involvement of reactive oxygen species and arachidonic acid in alpha 1-adrenoceptor-induced smooth muscle cell proliferation and migration. British Journal of Pharmacology 121: 665-670.
Owens GK (1995) Regulation of differentiation of vascular smooth muscle cells. Physiological Reviews 75: 487-517.
Patterson C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, Horaist C, Ballinger CA, Brasier AR, Bode C and Runge MS (1999) Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo. Journal of Biological Chemistry 274: 19814-19822.
Rosenfeld ME (1991) Oxidized LDL affects multiple atherogenic cellular responses. Circulation 83: 2137-2140.
Ross R (1993) The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362: 801-809.
Ross R (1999) Atherosclerosis─an inflammatory disease. The New England Journal of Medicine 340: 115-126.
Schwartz SM, Campbell GR and Campbell JH (1986) Replication of smooth muscle cells in vascular disease. Circulation Research 58: 427-444.
Segal AW and Abo A (1993) The biochemical basis of the NADPH oxidase of phagocytes. Trends in Biochemical Sciences 18: 43-47.
Sorescu D, Szocs K and Griendling KK (2001) NAD(P)H oxidases and their relevance to atherosclerosis. Trends in Cardiovascular Medicine 11: 124-131.
Steinberg D and Witztum JL. (2002) Is the oxidative modification hypothesis relevant to human atherosclerosis? Do the antioxidant trials conducted the date refute the hypothesis? Circulation 105: 2107-2111.
Sundaresan M, Yu ZX, Ferrans VJ, Irani K and Finkel T (1995) Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270: 296-299.
Suzuki YJ, Forman HJ and Sevanian A (1997) Oxidants as stimulators of signal transduction. Free Radical Biology & Medicine 22: 269-285.
Thyberg J, Blomgren K, Hedin U and Dryjski M (1995) Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study. Cell and Tissue Research 281: 421-433.
Thyberg J (1998) Phenotypic modulation of smooth muscle cells during formation of neointimal thickening following vascular injury. Histology & Histopathology 13: 871-891.
Wang Z, Castresana MR and Newman WH (2001) Reactive oxygen and NF-kappa B in VEGF-induced migration of human vascular smooth muscle cells. Biochemical and Biophysical Research Communications 285: 669-674.
Wedgwood S, Dettman RW and Black SM (2001) ET-1 stimulates pulmonary arterial smooth muscle cell proliferation via induction of reactive oxygen species. American Journal of Physiology 281: L1058-L1067.
Wu YJ, Hong CY, Lin SJ, Wu P and Shiao MS (1998) Increase of vitamin E content in LDL and reduction of atherosclerosis in cholesterol-fed rabbits by a water-soluble antioxidant-rich fraction of Salvia miltiorrhiza. Arteriosclerosis, Thrombosis & Vascular Biology 18: 481-486.
Yamboliev IA and Gerthoffer WT (2001) Modulatory role of ERK MAPK-caldesmon pathway in PDGF-stimulated migration of cultured pulmonary artery SMCs. American Journal of Physiology 280: C1680-C6188.
Zhou XM, Lu ZY and Wang DW (1996) Experimental study of Salvia miltiorrhiza on prevention of restenosis after angioplasty. Zhongguo Zhong Xi Yi Jie He Za Zhi. 16:480-482.
顏正華 (1991) 中藥學(上)，知音出版社，551-556.
顏正華 (1991) 中藥學(下)，知音出版社，320-323.
立得中醫研究室編著 (1992) 活血化瘀的研究，立得出版社，417-425.
陳可冀、史載祥主編 (1999) 實用血瘀証學，120-129