臺灣博碩士論文加值系統

大腸癌是一種常見的癌症，在台灣地區雖然大腸直腸癌位居癌症死因的第三位。但近年來的統計數據顯示每年也有將近新增病例約7000例，而其中約半數會因此死亡。
蓼科植物中的「藥用大黃」（Rheum officinale Baill）是一種常見的中醫臨床用藥，因具有強效的抗菌能力，過去也曾用來治療細菌性的痢疾、細菌性傷寒、霍亂……等疾病。而這種藥物所含的羥基蒽醌(hydroxyanthraquinones)是其主要的活性物質。在所屬羥基蒽醌的活性化合物內，包含五種相似化合物chrysophanol、emodin、 physcion、aloeemodin、rhein及它們所擁有的配醣物glucosides。
同屬蒽醌類化合物，且具有生物活性的蘆會大黃素Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone)，原取自大黃根莖部。實驗中發現，這種化合物能有效的抑制腫瘤增生以及誘導細胞凋亡的作用。然而目前對於蘆薈大黃素在人類大腸癌細胞株上，是否有生長抑制的能力及其機轉尚未明瞭，因此本實驗目的爲探討蘆薈大黃素誘導人類大腸癌細胞株細胞凋亡之分子機轉調控過程。
在我們的實驗結果中發現，大腸癌細胞給予蘆薈大黃素10、20、30、40及50 μM處理後，結果發現蘆薈大黃素對於細胞毒性的產生，具有時間上及劑量上影響。而蘆薈大黃素是以誘導細胞凋亡的方式使得細胞死亡。我們也發現當給予蘆薈大黃素時，會增加細胞內自由基的產生，以及造成細胞的氧化傷害。而在誘導細胞凋亡路徑探討中也證實，蘆薈大黃素會促使粒線體膜上的apoptosis inducing factor (AIF)、endonuclease G釋放出來，並且會造成DNA的斷裂。同時在外因性的凋亡路徑中，也發現細胞膜上的Fas/FasL表現量會增加，以及在下游的Caspase 8與Caspase 3也隨之被活化，使得細胞走向凋亡。
目前研究指出，基質金屬蛋白水解酶matrix metalloproteinases (MMPs)是一種蛋白水解酶，其作用會將細胞基質extracellular matrix （ECM）降解。細胞基質的降解，在癌細胞轉移侵襲上影響扮演著舉足輕重的角色。在我們的研究結果中亦指出，蘆薈大黃素可經由抑制MMP-2與MMP-9的表現進而抑制細胞的轉移。由上述結果可知蘆薈大黃素對於人類大腸癌細胞抗癌活性的影響機制，以及提供一個對未來更深入研究探討的基石，並開拓ㄧ新藥物發展為大腸癌臨床上治療與預防上的新契機。

Colorectal cancer is the most incident cancer and the third most common cause of cancer death in Taiwan, with approximately 7000 new cases and half number of deaths per year.
Rheum officinale Baill (Dahuang in Chinese), one of the important herb of traditional Chinese medicine. It has a strong antibacterial ability and had been used for the bacterial dysentery. The major active components of this herb are hydroxyanthraquinones, including chrysophanol, emodin, physcion, aloeemodin and rhein and their glucosides.
Aloe-emodin (1,8-dihydroxy -3-(hydroxymethyl)-anthraquinone) is a bioactive anthraquinone from the root and rhizome of Rheum palmatum. This compound has highly active in suppressing the proliferation of several cancer cell lines and induces apoptotic responses. However, it is not clear whether Aloe-emodin can induce growth inhibition of human colon cancer cells (colo 205) or not.
In our studies, the treatment of colo 205 cells with 10,20,30,40 or 50 μM Aloe-emodin will decerased the cell viability in a dose- and time-dependent manner. Aloe-emodin caused cell death by inducing of apoptosis. When cells were treated by Aloe-emodin, the value of intracellular reactive oxygen species and DNA damage was increased.
In our results suggest that Aloe-emodin induced cells apoptosis by releasing apoptosis inducing factor (AIF), endonuclease G from mitochondria, then caused DNA fragmentation. It also increased the expression of Fas/FasL, Caspase 8 and Caspase 3 cleavaging in examined cells.
In the present study, Matrix metalloproteinases (MMPs), one of the family member of closely related enzymes that degrade the extracellular matrix (ECM), are considered to play an important role of facilitating tumor invasion and spread. We have demonstrated that Aloe-emodin inhibits the invasion of human colon cancer cells by suppressing MMP-2 and MMP-9 expressions. These results indicate that a new mechanism of Aloe-emodin-mediated anticancer activity and it offered a justice for further investigation of Aloe-emodin as a therapeutic and preventive agent for human colon cancer in the future.

總目錄……………………………………………………………………1
表目錄……………………………………………………………………4
圖目錄……………………………………………………………………5
略字表……………………………………………………………………8
致謝………………………………………………………………………9
中文摘要…………………………………………………………………10
英文摘要…………………………………………………………………12
第一章 緒論……………………………………………………………14
第一節 大腸癌……………………………………………………14
第二節 Aloe-emodin藥理介紹……………………………………21
第三節 何謂細胞凋亡…………………………………………………24
第四節 氧化壓力與抗氧化防禦系統…………………………………34
第五節 細胞轉移與侵襲…………………………………………38
第六節 實驗目的設計及流程……………………………………41
第二章 實驗材料與方法………………………………………………43
第一節 實驗材料…………………………………………………43
第二節 實驗方法…………………………………………………50
壹 藥品配製…………………………………………………50
貳 冷凍細胞活化……………………………………………50
叁 細胞冷凍保存……………………………………………51
肆 人類大腸癌細胞培養……………………………………51
伍 細胞計數及存活測試……………………………………52
陸 檢測Aloe-emodin對細胞之增生與影響…………………54
柒 顯微鏡觀測Aole-emodin對細胞株的細胞形態之影響…55
捌 流式細胞儀檢測Aloe-emodin造成細胞凋亡情形………55
玖 西方點墨法檢測細胞內蛋白表現………………………56
拾 用洋菜糖膠體電泳 (agarose gel electrophoresis)檢測細胞DNA斷裂(DNA fragmentation)之影響…………………………62
拾壹 用單細胞凝膠電泳（Comet assay）檢測細胞DNA damage的影響…………………………………………………………………64
拾貳 細胞內reactive oxygen species（ROS）產生之證明…66
拾參 細胞內超氧陰離子之測定…………………………………67
拾肆 細胞粒線體膜電位測定……………………………………67
拾伍 細胞內鈣離子釋出之測定…………………………………69
第三章 結果
第一節Aloe-emodin對大腸癌細胞株（colo 205）形態之影響…………………………………………………………………………71
第二節 Aloe-emodin對細胞株大腸癌細胞株（colo 205）及平滑肌細胞（A10）之增生影響……………………………………………74
第三節 Aloe-emodin對細胞株colo 205之DNA傷害情形………77
第四節 Aloe-emodin細胞內自由基的表現………………………80
第五節Aloe-emodin造成粒線體膜電位之影響…………………84
第六節 Aloe-emodin細胞內鈣離子釋放情形……………………86
第七節 Aloe-emodin誘導細胞凋亡路徑蛋白探討………………88
第八節 Aloe-emodin抑制細胞轉移及其相關蛋白表現………101
第四章 討論…………………………………………………………109
第五章 結論…………………………………………………………120
第六章 參考資料……………………………………………………124

http://www.doh.gov.tw/cht/index.aspx
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