Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22622
標題: 利用差異性甲基化基因晶片分析黃麴毒素處理之人類肝細胞株之基因體甲基修飾變化
Analysis of Epigenetic Pattern Change of Aflatoxin B1-treated human liver cell line using differential methylation hybridization microarray
作者: 林冠伶
Lin, Kaun-Ling
關鍵字: 肝癌;hepatocellular carcinoma (HCC);黃麴毒素;甲基化差異基因晶片;aflatoxin B1 (AFB1);differential methylation hybridization (DMH) microarray
出版社: 生命科學系所
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摘要: 
肝癌是在全球人口統計中發生最為頻繁的癌症之一,其死亡率在全世界癌症高居第三,在台灣更分別為男、女癌症死亡之第一、二名。肝癌的發生成因眾多,包括有病毒感染(如HBV和HCV)及環境致癌物質造成基因變異,另外在基因上遺傳層次方面,也有為數眾多之證據顯示甲基化變異與肝癌相關。目前已有研究指出在肝癌病患檢體中,偵測到具高致癌性之環境致癌物質黃麴毒素(aflatoxin B1, AFB1)與DNA鍵結物(AFB1-DNA adducts)的發生,與解毒酵素GSTP1(Glutathione S-transferase)基因上游啟動子區段的高度甲基化具有顯著相關性。啟動子的高度甲基化會藉由不易與轉錄因子結合而降低基因的表現量,然而環境致癌物質和基因甲基化的直接相關性,其分子機制仍不明朗。本實驗選取八株肝癌細胞株(HA22T/VGH、HA59T/VGH、HepG2、Hep3B、HuH7、Mahlav、SK-hep-1與PLC/PRF/5),分別以RT-PCR檢測其第一階段解毒酵素細胞色素P450 (Cytochrome P450, CYP)、第二階段解毒酵素榖胱胺硫轉移酵素GST及腫瘤抑制基因p53之表現,並且以限制酵素Hea III檢測黃麴毒素攻擊是否導致p53之熱點突變。八株細胞株中,HuH7表現正常解毒酵素同時具有非點突變之原始型p53抑癌基因,為較趨於正常的肝細胞株,因此設計以HuH7細胞為實驗平台。在低血清培養下,以黃麴毒素處理HuH7肝細胞株,以24小時為間隔檢測其受到致癌物質攻擊前後之基因及上遺傳性(epigenetics)的整體變異。經由RT-PCR偵測到CYP、GSTP1確實受到黃麴毒素影響其表現量後,以甲基化差異基因晶片分析全基因體甲基化變化,結果顯示在處理時間點24小時、48小時、72小時及96小時後,處理組(以Cy5螢光標定)相對應於對照組(以Cy3螢光標定)細胞呈現相對高度甲基化的基因位點(Cy5/Cy3≧2.0)序列位點為265、479、579、498個,而相對低度甲基化則是96、16、29、34個。根據晶片的結果將針對有特別變化之基因設計表現量偵測及啟動子甲基化偵測的引子,藉以傳統分析法驗證黃麴毒素對於肝細胞的全面基因及基因上遺傳之影響。最後以生物反應路徑資料庫暨分析平台(ingenuity pathway analysis, IPA)分析甲基化具變異之點間生化路徑之關連性。
URI: http://hdl.handle.net/11455/22622
其他識別: U0005-0701200901515500
Appears in Collections:生命科學系所

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