左金丸抗肝癌效應及機轉分析

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Title:	左金丸抗肝癌效應及機轉分析 The anticancer effect and mechanism of Zuo-Jin-Wan and its alkaloidal ingredients in vitro and in mice
Authors:	周順頂
Contributors:	中醫學系碩士班
Keywords:	肝癌;中國醫藥;左金丸;HepG2細胞;肝內注射;c-myc hepatocellular carcinoma;traditional Chinese medicine;Zuo-Jin-Wan;human hepatoma HepG2 cells;intrahepatic injection;c-myc
Date:	2011-07-20
Issue Date:	2011-10-17 16:06:38 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	肝細胞癌(簡稱肝癌)是全球排名第五位的癌症，亞洲地區包括台灣是肝癌高盛行區，長年來肝癌始終位居為台灣10大癌症死亡率率的前二名。肝癌的防治刻不容緩！然而現代醫學（西醫）對肝癌的治療存有甚多不足及限制之處。而中國醫藥之臨床應用已超過兩千年歷史，近年來更常被用為各種癌症病患的臨床輔助療法或替代療法，中醫藥之抗肝癌效用急需科學驗證，以消弭中醫藥在肝癌臨床應用的不正確定性。本研究的目的是運用分子生物學的科技，探究中醫藥之抗肝癌潛力，並依中醫病機學的理論，選擇適用於肝癌常見證型－「肝脾濕熱」證的名方－「左金丸」，為主要素材。經由體外細胞模型，及活體荷瘤小鼠模型探討左金丸與其成份之抗肝癌效益。並運用c DNA微陣列分析（Microarray）探討左金丸與其成份之抗肝癌機轉。體外部分以MTT assay測試中醫傳處方左金丸、與左金丸的組成單味中藥黃連、吳茱萸及它們的主要活性物質Berberine、Evodiamine對人類肝癌HepG2/NF-κB轉殖細胞（攜帶冷光報導基因）生長的抑制效應。結果發現Berberine、Evodiamine、黃連、吳茱萸以及其複方左金丸，在體外對HepG2細胞都具抑制效果，且與時間及劑量呈正相關。對HepG2細胞48小時暴露的半數致死濃度（Lethal Concentration 50 LC50）各為：左金丸為7.7 μg/mL、黃連為4.3 μg/mL、吳茱萸為320 μg/mL、Berberine為5.9 μg/mL（16 μM）、Evodiamine為0.3 μg/mL（1μM），亦即Evodiamine毒殺作用最強，黃連、Berberine、左金丸等三組次之，而吳茱萸組對HepG2細胞毒殺作用最小。運用cDNA microarray探討左金丸與其成份在LC50劑量下對HepG2細胞基因表現圖譜的影響；經由網路分析（Network analysis）發現是經由抑制c-myc訊息傳遞路徑而達到效果。結果發現左金丸與黃連、吳茱萸對HepG2細胞毒殺效應較接近，而有效成分則與Berberine、Evodiamine較具關連，顯示左金丸抗肝癌效應其活性可能與其主要活性成分Berberine、Evodiamine有關。在活體模型部分，為了建立與臨床病理相似度更高的動物模型，以探究左金丸對荷人肝癌小鼠之抗肝癌效果，選擇免疫功能正常品系的ICR小鼠，採肝內注射HepG2細胞的方法造模，造模後第三天經活體冷光造影確認小鼠體內冷光值穩定表達時；依各小鼠之冷光值分為左金丸投藥組及造模對照組、投藥組小鼠每天餵食左金丸科學中藥200 mg/Kg（相當於60 Kg成人劑量每天12 g），對照組餵食相同容積的二次蒸餾水，於連續投藥後第7天、14天及28天等三個時間點將小鼠犧牲，進行巨觀與微觀評估，取肝臟作冠狀切片以推估其瘤體與肝臟重量比，取肝臟切片進行組織病理評估，取血清作生化分析觀察其變化。結果發現左金丸可有效抑制腹水的形成，尤其是腹水量較多的實驗初期（連續用藥7 天及14 天兩時間點）；與對照組間具有統計差異P<0.05。左金丸可有效減少小鼠肝臟瘤重/肝重比，尤其在肝癌細胞大量增生的實驗初期（連續用藥7 天）P<0.01。而血清生化分析結果發現隨著肝內注射HepG2細胞造模時間的增加，對照組小鼠之GOT持續上升，而左金丸投藥組之GOT則相對改善，然而因小鼠個體差異無法呈現統計意義。結果證實左金丸對「荷人類肝癌」小鼠活體模型具明確的抗肝癌作用，主要表現在減少因肝癌細胞增生所造成的小鼠肝臟瘤重/肝重比率；及減少因肝癌細胞浸潤而破壞內皮系統所形成的腹水增加。綜合以上結果我們認為，左金丸在體外及活體模型皆有明確的抗肝癌效應，有潛力成為肝癌臨床的輔助或替代性的療法。 Hepatocellular carcinoma (HCC) is ranked fifth in cancer incidence worldwide. In Taiwan, HCC is one of the top two leading causes of cancer death. There is as yet no proven effective medical therapy for treating HCC. Traditional Chinese medicine has been used in cancer treatment or as an adjuvant therapy for cancer treatment for centuries, but there is little scientific evidence on its effectiveness. The aim of this study was to investigate the anticancer effects and molecular mechanisms of a famous formula for patterns of damp heat in liver and gallbladder commonly seen in liver cancer patients - Zuo-Jin-Wan, its components (Coptis chinensis and Evodia rutaecarpa), and its major alkaloidal ingredients (berberine and evodiamine) in human hepatoma HepG2 cells and tumor-bearing mice. In order to use bioluminescence imaging to predict the effect of Zuo-Jin-Wan on hepatic tumor burden in mice. HepG2 cells were stably transfected with a NF-κB luciferase reporter construct to obtain HepG2/NF-κB/luc cells. For the in vitro studies, the treatment of HepG2/NF-κB/luc cells with Zuo-Jin-Wan, Coptis chinensis, Evodia rutaecarpa, berberine, and evodiamine significantly inhibited cell proliferation in a dose- and time-dependent manner by a MTT assay. The values of TC50 after 48 hours treatment were 7.7, 4.3, 320, 5.9, and 0.3 μg/mL, respectively. Then, HepG2/NF-κB/luc cells were treated with Zuo-Jin-Wan, Coptis chinensis, Evodia rutaecarpa, berberine, and evodiamine for 48 hours at TC50 doses and the total RNA was collected for cDNA microarray analysis. Hierarchical cluster analysis revealed that Coptis chinensis shared a similar gene expression profile with Zuo-Jin-Wan, suggesting that Coptis chinensis may be the main component responsible for the anticancer effects of Zuo-Jin-Wan. Network analysis showed that c-myc played a central role in the network topology. For the in vivo studies, the HCC xenograft model in immunocompetent mice was established by direct intrahepatic injection of HepG2/NF-κB/luc cells into ICR mice. Three days after tumor cell implantation, in vivo bioluminescence imaging in mice was performed and mice were assigned into the treatment group (Zuo-Jin-Wan, 200 mg/kg, gavage) and PBS control group. At varying time intervals of 7, 14, or 28 days after daily treatment with Zuo-Jin-Wan or PBS, mice were imaged and immediately sacrificed to examine therapeutic effects of Zuo-Jin-Wan. At 7 days after treatment, statistically significant decreases of tumor ascites fluid and the ratio of tumor weight to liver weight were observed in the Zuo-Jin-Wan group mice compared to control mice. The serum GOT levels in the Zuo-Jin-Wan group were decreased at 7, 14, or 28 days after treatment compared to control mice; however, there was no significant difference. In conclusion, Zuo-Jin-Wan, its components (Coptis chinensis and Evodia rutaecarpa), and its major alkaloidal ingredients (berberine and evodiamine) significantly suppressed tumor growth in HepG2 cells in vitro and in HepG2 tumor-bearing mice. Moreover, c-myc plays a critical role in the anticancer effect of Zuo-Jin-Wan. Our data not only provide scientific evidence for the anticancer effects of Zuo-Jin-Wan on liver cancer but also provide useful guidelines to Traditional Chinese Medicine doctors.
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