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研究生:徐依鈴
研究生(外文):I-Ling Hsu
論文名稱:鯽魚膽及虎耳草對內皮細胞增生的抑制作用
論文名稱(外文):The inhibitory effects of the aqueous extract of Pluchea indica or Saxifraga stolonifera Meerburg on proliferation of endothelial cell
指導教授:林銘德林銘德引用關係
指導教授(外文):Ming T. Lin
學位類別:碩士
校院名稱:慈濟大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:58
中文關鍵詞:鯽魚膽虎耳草內皮細胞
外文關鍵詞:Pluchea indicaSaxifraga stolonifera Meerburgendothelial cell
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中草藥在中國已流傳數千年,常用作為維持健康,預防及治療慢性病,扮演相當重要的角色。最近的研究顯示中草藥常具有多種生物活性,例如抗菌、抗病毒、抗黴、抗腫瘤、增強免疫力等效果。中草藥變成為許多疾病另一種治療的選擇。
具有促進血管新生的中草藥可用於治療心肌梗塞; 而具有抑制血管新生的中草藥則可用於治療腫瘤。為了要更進一步了解中草藥在血管新生過程中扮演的角色,過去幾年我們實驗室已篩選了288種中草藥,並根據其抑制血管新生的活性進行分類。在這其中我們挑選了17種較有較強抑制內皮細胞增生的中草藥,去研究它們對於細胞存活訊息中相當重要的分子Focal adhesion kinase (FAK)進行研究。在從這17種中草藥中挑選出兩種中草藥,分別為鯽魚膽 (Pluchea indica)與虎耳草 (Saxifraga stolonifera)進行之後的實驗。結果發現鯽魚膽與虎耳草除了能抑制FAK的磷酸化外,也會引起內皮細胞死亡,細胞死亡程度與中草藥濃度成正相關。但鯽魚膽與虎耳草所引起的細胞死亡是與caspase無關的。此外,也發現到鯽魚膽會降低內皮細胞的cdc2活化及cylin B1表現量下降,造成細胞週期被抑制在G2/M期。
我們也拿了另一個血管新生抑制因子: 血纖維溶脢原片段K4418與中草藥做比較。結果發現血纖維溶脢原片段K4418也會造成內皮細胞凋亡,並且也能造成FAK磷酸化下降以及FAK分解增加。
Chinese herbs have been used in China for hundreds of years as important remedies for maintaining health, prevention and treatment of chronic diseases. Recent studies have shown that extracts of Chinese herbs possess many biological activities including antibacterial, antiviral, antifungal, anti-tumor and immune-potentiation activities. Treatment with Chinese herbs for various diseases has become an important choice for alternative medicine.
Herbs with angiogenic activity can be used to rescue the myocardial ischemia and herbs with anti-angiogenic activity can be used to treat tumor. To understand the function of Chinese herbs in angiogenesis, in past year, our lab have screened 288 herbs for their anti-angiogenesis activity. Among them, we selected 17 Chinese herbs that had anti-proliferation ability to investigate their effects on focal adhesion kinase, which is activated for cell survival. Then we further picked 2 Chinese herbs, Pluchea indica and Saxifraga stolonifera, to investigate what molecules are involved in the inhibition of endothelial cell proliferation. We found that Pluchea indica and Saxifraga stolonifera induced endothelial cell death in a dose-dependent manner through caspase-independent pathway. In addition, Pluchea indica induced endothelial cell cycle arrest at G2/M phase through decresing cdc2 activation and cyclin B1 expression
Another antiangiogenesis factor, plasminogen fragment K4418 was also studied for comparison. K4418 induced endothelial cell apoptosis through caspase-dependent pathway. K4418 also decreased FAK phosphorylation and increased FAK degradation.
總目錄……………………………………………………………………….. Ⅰ
中文摘要…………………………………………………………………….. Ⅲ
英文摘要…………………………………………………………………….. Ⅳ
圖目錄……………………………………………………………………….. Ⅴ
縮寫檢索表 Ⅵ
緒論………………………………………………………………………….. 1
實驗方法…………………………………………………………………….. 6
A. 內皮細胞株繼代培養 6
 A-1 Calf pulmonary artery endothelial cells (CPAE)繼代培養 6
 A-2 細胞保存…………………………………………………………….. 6
 A-3 細胞解凍…………………………………………………………….. 7
 A-4 細胞計數…………………………………………………………….. 7
B. 細胞毒性性分析………………………………… 8
C. 細胞凋亡及其訊息傳導測定……………………………………….. 8
 C-1 細胞凋亡分析…………………………….. 8
 C-2 細胞凋亡相關因子之觀察……………………………….. 9
C-2a 細胞內蛋白質�vTotal cell lysates胝萃取…………………………… 9
C-2b 粒線體及細胞質之蛋白質萃取 9
D. 蛋白質分析……………………………………………………………. 10
 D-1 SDS-PAGE蛋白質電泳……………………………………………… 11
  D-1a SDS-PAGE膠體電泳製作……………………………………….. 11
  D-1b SDS-PAGE膠體電泳……………………………………….. 11
 D-2 西方點墨法…………………………………………………………. 11
 D-3 蛋白質定量…………………………………………………………. 12
E. Caspase活性分析………………………………………. 13
F. 細胞週期及其訊息傳導測定……………………………………………. 13
 F-1 細胞週期分析…………………………………………… 14
 F-2 細胞凋亡相關因子之觀察…………… 14
 F-2a 細胞內蛋白質�vTotal cell lysates胝萃取…………………………… 14
G. 血纖維溶�#鴘漯竁F與純化………………………………………. 15
 G-1 轉殖株大量蛋白質表現……………………………………….. 15
 G-2 重組蛋白的純化……………………………………... 16
 G-3 重組蛋白的濃縮…………………………………………... 16
H 統計方法………………………………………………............................. 17
結果……………………………………………….......................................... 18
 一、P. indica與S. Stolonifera引起之牛肺動脈內皮細胞凋亡.......................... 18
 二、P. indica與S. Stolonifera引起牛肺動脈內皮細胞凋亡之訊息傳遞路徑 19
 三、P. indica引起牛肺動脈內皮細胞之細胞週期抑制... 20
 四、P. indica引起牛肺動脈內皮細胞之細胞週期抑制 21
五、P. indica引起FAK與其下游Akt蛋白酪氨酸磷酸化的改變 21
 六、血纖維溶�#鴙垓梜J白K4418引起之牛肺動脈內皮細胞凋亡 21
 七、K4418降低bFGF及VEGF引起之蛋白酪氨酸磷酸化..................... 22
八、K4418造成FAK磷酸化下降及FAK分解................ 22
討論............................ .... 23
結論................................................ ................................................ ................ 27
參考文獻................................................ ................................................ ........ 28
圖................................................ ................................................ .................... 33
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