臺灣博碩士論文加值系統

動脈粥狀硬化(Atherosclerosis)是心血管疾病中主要的疾病，在動脈粥狀病變與血管再窄化發展病程中，血管平滑肌細胞從中膜層遷移至新生血管內膜是血管形成病變的重要的因素。在血管受傷病變過程中，平滑肌細胞會由收縮態轉變為合成態，並由血管中層往內膜層增生和遷移，導致血管內膜增厚。樟芝(Antrodia camphorata，AC)是一種中國傳統常見的藥品，它具有抑制腫瘤細胞增生及遷移的作用。此論文欲研究樟芝對大鼠動脈平滑肌細胞 (Rat aortic smooth muscle cell；RASMC：A7r5)的增生及遷移是否也具有抑制效果；並且評估其對老鼠頸動脈受傷後之新生內膜生成的影響。MTT和BrdU 增生實驗主要是研究樟芝對平滑肌細胞增生作用的影響，由MTT和BrdU 實驗結果皆顯示樟芝可抑制血小板衍生生長因子(PDGF)所引起平滑肌細胞的增生作用。在Transwell 遷移實驗中，大鼠動脈平滑肌細胞(RASMC)先用樟芝萃取液或食鹽水處理後，然後血小板衍生生長因子(PDGF)加在Transwell的下層刺激細胞遷移，計算由上層遷移到下層的細胞數目。在wound scratch assay中，大鼠動脈平滑肌細胞(RASMC)用樟芝萃取液或食鹽水預先處理，使用pipette tips 刮除細胞；然後用血小板衍生生長因子(PDGF)刺激並培養24小時。此2個實驗結果皆顯示樟芝可顯著抑制血小板衍生生長因子(PDGF)所引起的平滑肌細胞遷移作用。動物實驗方面，將C57BL/6老鼠的左邊頸動脈在靠近分支處結紮手術，術後的老鼠用水及樟芝萃取液連續灌食4星期，新生內膜生成的嚴重性則用新生內膜/中層(neointima / media，N/M )比例來表示。在4星期後，以樟芝萃取液治療，經頸動脈結紮的老鼠其新生內膜生成較少(N/M ratio,水控制組, n=13 vs 樟芝 250 mg/kg, n=12 vs 樟芝1250 mg/kg, n=12；1.33±0.87 vs 0.83±0.45vs 0.63±0.32，P < 0.05)。實驗結果顯示樟芝對於血小板衍生生長因子(PDGF)引起平滑肌細胞的增生和遷移作用是個有效的抑制劑，另外以樟芝治療也可降低老鼠頸動脈結紮手術後的新生內膜的生成。

Atherosclerosis in western countries and Taiwan is the major cardiovascular diseases. The migration of vascular smooth muscle cells (VSMCs) from the media into the neointima is a key event in the development of atherosclerotic lesion and in restenosis. An important hallmark of vascular injury is the increased proliferation and migration of vascular smooth muscle cells (VSMCs) associated with the transformation from contractile to synthetic phenotype. Antrodia camphorate (AC) is a well-known traditional Chinese medicine. It has been shown to inhibit the proliferation and migration of cancer cells. This study examined whether AC could inhibit rat aortic smooth muscle cell (RASMC：A7r5) proliferation and migration and evaluated its effect on neointima formation in mouse carotid artery after injury. MTT and BrdU proliferation assay were used to evaluate AC effect on SMC proliferation. Both assays showed AC inhibits PDGF-induced SMC proliferation. In Transwell migration assay, RASMCs were treated with AC or saline. PDGF (10 ng/ml) was added to the lower compartment. Migrated cells on the lower side were fixed and counted. In wound scratch assay, RASMCs were pretreated with AC or saline and were seeded into confluent cell monolayers. They were wounded with pipette tips and stimulated with 10 ng/ml PDGF for 24 hrs. Both assays also showed AC could significantly inhibit PDGF-induced SMC migration. The left common carotid arteries of C57BL/6 mices were ligated near the carotid bifurcation. The mice were fed with water or AC for 4 wks. The severity of neointima was expressed as neointima/media (N/M) ratio. The AC-treated mice had less neointima formation at 4 wks after carotid ligation (N/M ratio, water, n=13 vs 250 mg/kg AC, n=12 vs 1250 mg/kg AC, n=12；1.33±0.87 vs 0.83±0.45 vs 0.63±0.32, P < 0.05). The data indicate that AC is an effective inhibitor of PDGF-induced RASMC proliferation and migration. AC treatment reduced neointima formation in mouse carotid ligation model.

中文摘要---------------------------------------------------------------------------------------- 1
英文摘要---------------------------------------------------------------------------------------- 2
誌謝---------------------------------------------------------------------------------------------- 4
目錄---------------------------------------------------------------------------------------------- 5
圖目錄------------------------------------------------------------------------------------------- 7
表目錄--------------------------------------------------------------------------------------------9
附錄-----------------------------------------------------------------------------------------------9
縮寫表------------------------------------------------------------------------------------------- 10
第一章 緒論------------------------------------------------------------------------------------ 11
第二章 研究動機與目的--------------------------------------------------------------------- 24
第三章 材料與方法--------------------------------------------------------------------------- 26
3-1 實驗材料----------------------------------------------------------------------------------- 27
3-2 實驗藥品----------------------------------------------------------------------------------- 27
3-3 實驗儀器----------------------------------------------------------------------------------- 29
3-4 實驗方法----------------------------------------------------------------------------------- 30
3-4-1 細胞存活率測定(MTT assay)------------------------------------------------------- 31
3-4-2 細胞存活率測定(BrdU assay) ----------------------------------------------------- 32
3-4-3 細胞遷移實驗(cell migration assay) -------------------------------------------- 32
3-4-4 細胞刮傷試驗(Wound scratching assay) -------------------------------------- 33
3-4-5 Gelatin Zymography----------------------------------------------------------------- 33
3-4-6 動物實驗(Animal test)--------------------------------------------------------------- 37
3-4-7 組織切片及染色----------------------------------------------------------------------- 38
3-4-8 血管檢查評估-------------------------------------------------------------------------- 39
3-4-9 統計分析法----------------------------------------------------------------------------- 40
第四章 實驗結果------------------------------------------------------------------------------ 41
第五章 討論------------------------------------------------------------------------------------ 44
第六章 參考文獻------------------------------------------------------------------------------ 47
圖表---------------------------------------------------------------------------------------------- 51
表格---------------------------------------------------------------------------------------------- 68
附錄---------------------------------------------------------------------------------------------- 69
自述---------------------------------------------------------------------------------------------- 75

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