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研究生:蔡佩玲
研究生(外文):Pei-Lin
論文名稱:七層塔水萃物誘發人類肺腺癌細胞株 A549細胞凋亡之分子機制探討
論文名稱(外文):Investigation of Aqueous O. gratissimum extract-induced A549 human lung carcinoma cells apoptosis
指導教授:高紹軒
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生化暨生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:73
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惡性腫瘤自1982年起,即列為國人死因之首位。其中,肺癌是目前國人十大癌症死亡的第一位,尤以肺腺癌患者居多,佔所有肺癌類型的40 %,肺癌在治療上十分不易且復發率高,在傳統西醫的治療方法中,不管哪一種方式,皆會產生令人不適的副作用。然而,中草藥的研究在近年來成為十分熱門的研究主流,因為其副作用極低,來源天然無害且效果良好。七層塔屬於羅勒的一種,是台灣特有的的民間草藥,其功效為抗氧化、抗癌、抗肝臟纖維化等,具有極高之藥用價值,目前尚未有七層塔對於人類肺腺癌細胞(A549)抑制機轉之相關研究,因此,本研究利用七層塔之水萃物刺激人類肺腺癌細胞株A549。結果發現,七層塔水萃物能有效抑制細胞存活率,具有dose-dependent的現象。進一步以蛋白質分析試驗,發現七層塔會抑制Bcl-2蛋白表現並透過活化內生性粒腺體路徑caspase-9和外生性路徑caspase-8,進一步活化下游因子caspase-3而引起細胞凋亡。在MAPK訊息分析結果也發現,A549細胞在七層塔處理之後,JNK、p38表現上升,ERK表現則下降。顯示七層塔會藉由JNK以及p38路徑來促使細胞進行凋亡。再以二維式電泳分析來探討蛋白質的表現,結果顯示有14個蛋白質表現顯著上升,以MALDI-MS鑑定之後,發現這些蛋白質,包含Heat shock protein、Peroxiredoxin-1、Triosephosphate isomerase、NADH dehydrogenase 、Actin、SLC15A3 protein、Retinal dehydrogenase 1、Malignant T cell amplified sequence 1等。本實驗也進一步以銀染色來探討更多蛋白質的變化,未來可以深入探討這些點所代表之蛋白質為何,是否與肺癌之間具有相關性,或許可做為有潛力的生化標記。

Since 1982, cancer is the leading cause of deaths in Taiwanese and lung cancer contributes the main death among the cancers. Approximately 40% of lung cancers are adenocarcinomas, and the others are 30 % of Squamous cell carcinoma and 10% of large cell carcinoma and 20% of Small cell carcinoma.In clinic, lung cancer is hard to cure and control with the current treatments, such as surgery resection, chemotherapy and radiotherapy, which frequently cause undesirable and adverse side effects. Recently, mounting evidences reveal that use of medicinal plants may be regarded as a supplement with good efficacy and few side effects for the current cancer treatment. Among the medicinal plants, Ocimum gratissimum Linn has been extensively used in Taiwan. Ocimum gratissimum Linn has been reported to show anti-oxidant, anti-carcinogenic and anti-fibrosis therapeutic effects. However, the underlying antitumor mechanism of Ocimum gratissimum Linn against lung cancer still remains unclear. Here we aimed to investigate the effects of aqueous extract of OG leaf (OGE) on malignant lung cancer cell line A549. Our results revealed that OGE dose -dependently decreased the cell viability of A549. Further investigation showed that OGE suppressed the expression of Bcl-2 and the activation of caspase-9, caspase-8 and caspase-3, which may synergistically induce the apoptosis of A549. Additionally, OGE treatment was found to increase the phosphorylation of p38 and JNK but decrease the phosphorylation of ERK. Proteomic approach, using two-dimensional gel electrophoresis (2-DE) and MALDI-MS analysis, revealed that OGE treatment led to a differential protein expression profile as comparing with mock control. Among the protein spots with differential expression on 2-D gel, 14 spots were further in-gel digested and identified by peptide-mass fingerprint (PMF). The identified proteins including Heat shock protein, peroxiredoxin-1, triosephosphate isomerase, NADH dehydrogenase, actin, SLC15A3 protein, retinal dehydrogenase 1 and malignant T cell amplified sequence 1. In conclusion, our findings indicate that OGE effectively induce the apoptosis of A549 cell through the mitochondrial apoptotic pathway and the activation of p38 and JNK mitogen-activated kinase pathway. Further proteomic analysis reveals that OGE treatment results in a different protein expression pattern; however, the proteins showing differential level still need further investigation and biological validation for mining potential biomarkers.

目錄………………………………………………………………..2
誌謝……………………………………………………..........3
中英文摘要………………………………………………………..5
縮寫表……………………...…………………………………...9
壹、緒論……………..…………………………………………..10
貳、研究動機與目的……………………………………………..19
參、實驗架構…………………………......................20
肆、實驗材料及方法……………………………………………..21
伍、實驗結果………………………………………............34
陸、討論…………………………………………………………..39
柒、參考文獻………………………………………………………43
捌、圖表與圖表說明………………………………………………48
玖、附錄……………………………………………………………70


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