臺灣博碩士論文加值系統

腫瘤細胞中，血管內皮細胞生長因子（vascular endothelial growth factor，VEGF）與基質金屬蛋白酶（matrix metalloproteinase，MMP）抑制劑可藉由許多不同的訊息傳遞路徑，達到具有抑制血管新生及細胞增生的效果。過去對於中草藥調節血管新生的作用機轉較少被探討。本研究使用各種不同中草藥投至人類肝癌細胞（HepG2及Huh 7），先以細胞存活率試驗初步篩選具生長抑制的藥物。實驗結果顯示，香蘇散及黃連解毒湯為複方中最具效力的待測中草藥。香蘇散能夠降低VEGF的蛋白質表現，並可藉由抑制MMP-9的RNA與蛋白質表現以抑制其蛋白質活性；初步判斷，其有效單方紫蘇葉藉由抑制VEGF表現以及MMP-9的活性以降低肝癌細胞的侵犯與血管新生作用。
黃連解毒湯可以抑制β-catenin的蛋白質表現，其有效單方黃連，除了能抑制β-catenin蛋白質表現外也會調降MMP-9的蛋白質表現與活性。
另外，單方藥物：石上柏、夏枯草、鴨膽子、苦參與黃獨均能抑制肝癌細胞生長；其中，夏枯草與黃獨亦能抑制VEGF表現，而鴨膽子與苦參是藉由抑制MMP-9及β-catenin的蛋白質表現以抑制肝癌細胞的侵犯與血管新生作用。
本實驗以蛋白質體學的方法，篩選具有調節肝癌血管新生相關因子的中草藥，達到中草藥實證效應的目標。

目錄
中文摘要------------------------------------------------------------I
英文摘要-----------------------------------------------------------III
壹、 緒論------------------------------------------------------------1
一、癌細胞增生、轉移與血管新生的關係------------------------------------1
二、影響血管新生的相關蛋白質------------------------------------------------3
三、中草藥研究的歷史沿革------------------------------------------------------6
四、研究目的-------------------------------------------------------------------------8
貳、 材料與方法---------------------------------------------------9
一、細胞培養-------------------------------------------------------------------------9
二、Total RNA萃取------------------------------------------------------------------9
三、反轉錄聚合酵素鏈反應-----------------------------------------------------11
四、蛋白質之萃取及定量--------------------------------------------------------12
五、西方轉漬法（Western blot）偵測血管新生調節因子-----------------13
六、中草藥複方或單方樣品的來源--------------------------------------------15
七、細胞存活率分析（MTT assay）------------------------------------------15
八、半數抑制濃度計算（IC50）------------------------------------------------16
九、細胞侵犯能力試驗（Invasion assay）-----------------------------------16
十、明膠酶譜法（Gelatinase Zymography）---------------------------------17
十一、資料分析（Data analysis）---------------------------------------------18
參、 結果-----------------------------------------------------------19
一、中草藥複方對肝癌細胞增生的影響-------------------------------------19
二、中草藥複方及其有效單方對肝癌細胞增生與血管新生的影響----20
三、中草藥單方對肝癌細胞增生與血管新生的影響----------------------25
肆、 討論-----------------------------------------------------------31
伍、 未來展望-----------------------------------------------------40
陸、 參考文獻-----------------------------------------------------41


圖目錄
圖一、（A）小柴胡湯（水萃取）處理HepG2細胞株之生長曲線-----------49
圖一、（B）小柴胡湯（70%酒精萃取）處理HepG2細胞株之生長曲線-50
圖一、（C）柴胡加龍骨牡蠣湯處理HepG2細胞株之生長曲線-------------51
圖一、（D）核桃承氣湯處理HepG2細胞株之生長曲線------------------------52
圖一、（E）半夏厚朴湯處理HepG2細胞株之生長曲線------------------------53
圖一、（F）香蘇散處理HepG2細胞株之生長曲線------------------------------54
圖一、（G）黃連解毒湯處理HepG2細胞株後之生長曲線--------------------55
圖一、（H）紫蘇葉處理HepG2細胞株後之生長曲線--------------------------56
圖一、（I）黃連處理HepG2細胞株後之生長曲線------------------------------57
圖一、（J）石上柏處理HepG2細胞株後之生長曲線---------------------------58
圖一、（K）夏枯草處理HepG2細胞株後之生長曲線--------------------------59
圖一、（L）鴨膽子處理HepG2細胞株後之生長曲線--------------------------60
圖一、（M）山慈菇處理HepG2細胞株後之生長曲線-------------------------61
圖一、（N）苦參處理HepG2細胞株後之生長曲線-----------------------------62
圖一、（O）黃獨處理HepG2細胞株後之生長曲線-----------------------------63
圖二、（A）香蘇散處理Huh 7細胞株之生長曲線------------------------------64
圖二、（B）黃連解毒湯處理Huh 7細胞株後之生長曲線----------------------65
圖二、（C）紫蘇葉處理Huh 7細胞株後之生長曲線----------------------------66
圖二、（D）黃連處理Huh 7細胞株後之生長曲線------------------------------67
圖二、（E）石上柏處理Huh 7細胞株後之生長曲線---------------------------68
圖二、（F）夏枯草處理Huh 7細胞株後之生長曲線----------------------------69
圖二、（G）鴨膽子處理Huh 7細胞株後之生長曲線---------------------------70
圖二、（H）山慈菇處理Huh 7細胞株後之生長曲線---------------------------71
圖二、（I）苦參處理Huh 7細胞株後之生長曲線-------------------------------72
圖二、（J）黃獨處理Huh 7細胞株後之生長曲線------------------------------73
圖三、HepG2 細胞侵犯能力實驗結果------------------------------------------74
圖四、中藥單方對HepG2 細胞侵犯能力實驗結果---------------------------75
圖五、Huh 7 細胞侵犯能力實驗結果--------------------------------------------76
圖六、中藥複方處理HepG2之血管新生相關蛋白表現-----------------------77
圖七、中藥單方處理HepG2之血管新生相關蛋白表現---------------------78
圖八、複方及其有效單方處理Huh 7之血管新生相關蛋白表現------------79
圖九、中藥單方處理Huh 7之血管新生相關蛋白表現------------------------80
圖十、MMP-9 PCR偵測中藥複方對於HepG2之RNA表現-------------------81
圖十一、複方及其有效單方對於HepG2之MMP活性測試-------------------82
圖十二、鴨膽子與苦參對於HepG2之MMP活性測試----------------------83


表目錄
表一、中草藥複方萃取物處理HepG2之IC50------------------------------------84
表二、中草藥單方萃取物處理HepG2之IC50------------------------------------85
表三、中草藥萃取物處理Huh 7之IC50-------------------------------------------86
表四、中草藥萃取物對於肝癌細胞血管新生相關蛋白的影響--------------87
附錄一、草藥配方----------------------------------------------------------------88

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謝明村（1996）中藥學概論，中國古典醫籍新解叢書（台北市）

成佳憲，行政院衛生署，中醫藥委員會95年度計畫編號： CCMP95-RD-210中藥材黃連組成物對肺癌及肝癌放射線治療動物模式之免疫調控因子基因表現

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