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研究生:羅仁傑
研究生(外文):Ren-Jie Luo
論文名稱:牛樟芝中抑制血管新生活性成分之研究
論文名稱(外文):Anti-angiogenic activities of active components isolated from Antrodia Camphorata fruiting body
指導教授:吳宗正吳宗正引用關係
指導教授(外文):Tzong-Zeng Wu
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:91
中文關鍵詞:血管新生牛樟芝血管內皮細胞生長因子雞胚胎絨毛尿囊膜
外文關鍵詞:angiogenesischick chorioallantoic membraneAntrodia camphorataVascular endothelial growth factor
相關次數:
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  • 收藏至我的研究室書目清單書目收藏:1
血管新生 ( angiogenesis ) 是形容原先存在的血管或位於微血管後段小靜脈長出新生血管的過程。對於大多數實質腫瘤而言,血管新生對於腫瘤的生長與轉移是必要的。藉由抑制血管增生進一步抑制腫瘤的生長,為近年來相當熱門的研究課題。牛樟芝為台灣特有的藥用真菌,其已被證實具有抗癌、抗腫瘤的活性;本研究目的在於評估從牛樟芝子實體乙醇粗萃取物 ( ACCE )中利用管柱層析法所分離出來的兩個分離物 ( Fra-10, Fra-13 ),是否具有抑制血管新生的潛力。在內皮細胞存活率實驗中發現,以ACCE, Fra-10以及Fra-13處理24小時之後,細胞存活率隨著給藥濃度上升而下降 (12.5 -100 μg/ml) ,而內皮細胞對Fra-10較為敏感; 另外,實驗結果也發現Fra-10在濃度25 μg/ml之下,能夠抑制HUVEC的遷移以及微小管腔的形成 ; 在活體實驗部分,使用雞胚胎絨毛尿囊膜試驗以及鼠體內血管新生實驗,由雞胚胎絨毛尿囊膜實驗結果顯示 ,Fra-10在濃度1 mg/ml下,減少了血管新生的數目;最後,鼠體內血管新生實驗則發現,Fra-10在濃度200 μg/ml下也具有抑制血管新生效果。推測Fra-10可能會間接的影響血管內皮細胞生長因子VEGF (Vascular endothelial growth factor) ,造成內皮細胞的生長、遷移以及分化受到抑制,進而產生抑制血管增生的效果。



Angiogenesis is a process through which new blood vessels develop from pre-existing vessels. For generality of solid tumors, angiogenesis is essential for their growth and metastasis. Recently, anti-angiogenesis has become an attractive, potential strategy for cancer therapy. Antrodia camphorata (AC) is a kind of medical fungus that only occurring in Taiwan. It has been reported that extracts of AC possess antitumor activities. The aim of this study was designed to evaluate the anti-angiogenesis effect of AC ethanol crude extract (ACCE) and two fractions of isolated from the fruiting body of Antrodia camphorata. In this study, we demonstrated that fraction 10 (Fra-10) and fraction 13 (Fra-13), two AC partially purified extracts, at the concentration ranged from 12.5 to 100 μg/ml caused dose-dependent inhibition in human umbilical vein endothelial cells (HUVEC) growth, as measured by the MTT assay. In addition, Fra-10 inhibited cell migration and capillary-like tube formation in HUVECs at the concentration 25 μg/ml. An in vivo angiogenesis assay using chick chorioallantoic membrane (CAM) model, Fra-10 showed that decrease of the vascular density index (VDI) in concentration of 1 mg/ml. In Matrigel plug assay Fra-10 inhibited new blood vessel formation in Matrigel plugs implanted in C57BL/6 mice at the concentration 200 μg/ml. We speculated that Fra-10 might indirectly interact with VEGF, which inhibited the cell growth, migration and capillary-like tube formation of HUVECs, therefore inhibited the angiogenesis.



摘要……………I
Abstract……………III
目錄……………IV
圖目錄……………VI
表目錄……………VII
壹、 緒論……………1
一、 血管新生……………1
二、 腫瘤與血管新生……………1
三、 血管新生的調控……………3
四、 血管新生與癌症治療……………4
貳、 文獻回顧……………6
一、牛樟芝簡介……………6
(一)、植物學分類……………6
(二)、生長特徵……………9
二、牛樟芝成份分析……………10
(一) 一般成分……………10
(二) 生理活性成分……………11
三、牛樟芝生理活性功能……………17
四、牛樟芝的安全性……………18
參、研究目的……………21
肆、實驗材料與儀器……………23
一、實驗材料……………23
二、實驗耗材……………24
三、實驗儀器……………25
伍、實驗方法……………27
一、牛樟芝子實體萃取……………27
二、利用管柱層析法分離乙醇粗萃取物……………27
三、HPLC成分分析……………29
四、細胞培養與測試……………29
(一)、細胞培養……………29
(二)、細胞測試……………30
1. 細胞毒性測試……………31
2. 內皮細胞遷移能力測試……………31
3. 內皮細胞微小管腔形成能力測試……………32
五、雞胚胎絨毛尿囊膜測試……………33
1. 絨毛尿囊膜測試……………34
2. 絨毛尿囊膜測試中血管新生分析……………34
六、小鼠體內血管新生測試……………35
1. Matrigel plug assay……………35
2. Hemoglobin 含量測定……………36
陸、結果……………39
一、牛樟芝子實體乙醇粗萃取物分離純化……………39
二、細胞存活率測試……………39
三、內皮細胞遷移能力測試……………40
四、內皮細胞微小管腔形成能力測試……………41
五、雞胚胎絨毛尿囊膜測試……………42
六、小鼠體內血管新生測試……………43
柒、討論……………63
捌、未來展望……………67
參考文獻……………69
附錄1 牛樟芝生理活性功能……………81
附錄2 IC50計算……………89
附錄3 Hemoglobin標準品製作標準曲線……………91
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