臺灣博碩士論文加值系統

研究背景: 肝癌是在東方國家最常見到的一種惡性腫瘤，然而至今仍無十分有效的藥物用以治療這種疾病，有越來越多的證據顯示許多由中草藥分離出的天然物具有抗癌功效，利用傳統醫藥在預防或是治療癌症的研究報告也逐年增加。在癌症的治療中，首要目標就是選擇性的使癌細胞死亡。而細胞凋亡是一種普遍且有效率的細胞自殺途徑。許多研究顯示一些由中草藥當中萃取出的抗癌藥物其具備的抗癌活性乃是藉由誘發癌細胞進行細胞凋亡所達成。在本實驗中，我們製備了二十種中草藥的萃出物用來評估其抑制人類肝癌細胞生長的效應及探討其作用機制。
材料與方法: 在本研究中，我們以MTT細胞存活率測定方法來測定二十種中草藥抑制HepG2細胞的生長效應。並藉由DNA片段化的特徵以及一些細胞染色方法：Hoechst 33342、TUNEL、Annexin V-FITC等方法來辨別細胞死亡是經由細胞凋亡或是細胞壞死途徑。我們也利用Agilent human 1A oligo microarray（含22,575個基因）來偵測經由紫蘇萃出物處理的HepG2細胞其基因表現。
結果與討論: 紫蘇萃出物為二十種中草藥當中所偵測出最具抑制HepG2細胞生長效應的中草藥。我們並以豬的肝臟細胞來測試紫蘇萃出物對於正常細胞的細胞毒性，研究結果顯示與癌細胞相較之下，紫蘇萃出物對於非癌細胞所造成的細胞毒性很低。藉由DNA片段化的特徵以及一些細胞染色方法：Hoechst 33342、TUNEL、Annexin V-FITC等方法顯示出紫蘇萃出物抑制HepG2細胞生長的效應是藉由引發HepG2細胞進行細胞凋亡。我們也使用DNA microarray進一步分析與細胞凋亡相關的基因群。根據基因表現的分示結果，我們提出二個由紫蘇萃出物引發HepG2細胞凋亡的假設途徑：一是藉由活化由death receptor所調控的caspase cascade相關基因群，二是藉由活化由p53基因所調控的mitochondrial pathway。
結論: 我們建立了一套用來檢測具抗癌活性中草藥的體外實驗系統，此系統包含了細胞存活率測定、細胞凋亡檢測（DNA片段化檢測以及特殊的螢光染色方法）、以及DNA晶片分析。根據這項系統，我們成功的篩選出具有抗癌潛能的紫蘇萃出物。由DNA晶片的分析結果顯示紫蘇抗癌的活性乃在於其誘發癌細胞走向細胞凋亡所致。

Background: Hepatocellular carcinoma is one of the most common malignant tumors in oriental countries. However, effective drugs for treating this disease are still unavailable. Emerging evidence has demonstrated that many natural products isolated from plant sources possess antitumor properties. Interest in exploiting traditional medicines for prevention or treatment of cancer is increasing. In cancer treatment, the therapeutic goal is to trigger tumor-selective cell death. Apoptosis represents a universal and exquisitely efficient cellular suicide pathway. Therefore, several studies have shown that some of chemotherapeutic agents isolated from Chinese herbal extracts exert their anticancer activity by inducing cancer cells apoptosis. In the present study, 20 herbal extracts were prepared and used to evaluate the effect and mechanism of growth inhibition on human hepatoma cells (HepG2).
Materials and methods: In this study, MTT cell viability assay was used to measure the antiproliferative effects of 20 herbal extracts on HepG2 cells. Two cell death types, apoptosis and necrosis, were distinguished by DNA fragmentation detection and by several fluorescent staining such as Hoechst 33342, TUNEL assay, and Annexin V-FITC staining assay. Agilent human 1A oligo microarray containing 22,575 oligonucleotide probes was used to screen the differentially expressed genes in the HepG2 cells treated with Perilla frutescens extract.
Results and discussion: Among the 20 Chinese herbal extracts tested, P. frutescens showed the best efficiency in growth inhibition on HepG2 cells. The cytotoxicity of P. frutescens extract was also evaluated in porcine liver cells. Significantly lower cytotoxicity on such non-cancer cells was observed when compared with HepG2 cells. The results of DNA fragmentation detection and several fluorescent staining indicated that the P. frutescens extract could trigger the pathway of cellular apoptosis and resulting the cell death in HepG2 cells. The apoptosis related genes were further analyzed using DNA microarray. Based on the result of differentially expressed genes, we proposed two pathways in which P. frutescens extract induced HepG2 cells apoptosis. The first one was by activating the caspase cascade of death receptor related genes and the second one was by p53 activating the mitochondrial pathway.
Conclusion: In vitro assay system, including the cell viability assay, apoptosis determinations of DNA fragmentation, specifically fluorescent staining and DNA microarray analysis, has been established in screening potent anticancer extracts from Chinese herbs. According to the system, we successfully identified the potent anticancer activity of P. frutescens extract. The anticancer effect of P. frutescens extract was proved to be dependent on apoptosis as indicated by DNA microarray analysis.

CONTENTS
Abstract (in Chinese) 1
Abstract 3
Contents 5
Chapter 1
Background (Literatures review) 6
Chapter 2
High Performance Liquid Chromatography (HPLC) Analysis of Perilla frutescens Extract 21
Chapter 3
Effects of Chinese Herbal Extracts on the Growth Inhibition of HepG2 Cells and Primary Liver Cells 24
Chapter 4
Induction of Apoptosis by the Treatment of Perilla frutescens Extract on HepG2 Cells 37
Chapter 5
Differentiating Apoptosis Related Genes of HepG2 Cells Exposure to Perilla frutescens Extract Using Microarray Analysis 51
Chapter 6
Conclusion 63
References 66

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