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研究生:黃怡媛
研究生(外文):Yi-Yuan Huang
論文名稱:在人類細胞中建立合成致死篩選系統及其在抑癌基因PTEN研究之應用
論文名稱(外文):Establishing a Synthetic Lethal Screening System in Mammalian Cells and Its Application in Tumor Suppressor Gene PTEN
指導教授:林敬哲林敬哲引用關係
指導教授(外文):Jing-Jer Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:58
中文關鍵詞:合成致死
外文關鍵詞:PTENSynthetical lethalTep1
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合成致死(synthetic lethal)的意義是指當細胞只有單一個基因發生突變時,細胞可存活;而當細胞同時發生兩個基因的突變時,細胞就無法繼續存活,具有這種關係的基因,稱之為合成致死。在酵母菌中,合成致死的篩選應用已被建立完備,然而還未建立在人類細胞中。由於大部分的癌細胞源至於正常細胞的基因突變,因此利用合成致死篩選出當單獨存在時不會致死(即發生在正常細胞時),但若與癌細胞原本具有的遺傳缺失共同存在時就會致死的其他基因突變,因此,這些被篩選出的基因產物即可作為一個新的藥物作用目標,並且選擇性的殺死癌細胞。本論文主要目標為建立合成致死篩選(synthetic lethal screening)系統於人類細胞,於是我們使用 PTEN 突變的 H1299 細胞株,PTEN是人體重要的抑癌基因之一,在許多悪性腫瘤都具有高比率的突變現象。我們將帶有正常 PTEN 和 EGFP 基因的核外複製質體送入 H1299 細胞株,此質體的存在可藉由 EGFP 的表現偵測。當製造其他基因的突變和 PTEN 產生合成致死時,H1299 細胞株會保留帶有正常 PTEN 的核外複製質體以維持存活,因此我們可以藉由質體上 EGFP 的表現偵測出和 PTEN 產生合成致死的細胞。我們同時在酵母菌中篩選出和TEP1合成致死的基因,TEP1 是在酵母菌內中和 PTEN 同源且相似度最高的基因,再以RNA silencing的方式,來驗證其與PTEN合成致死的關係。藉由上述方法,我們希望能將整個合成致死篩選系統於人類細胞中建立完備,並且找到和 PTEN 合成致死的基因,作為抗癌藥物研發的新方向。
Synthetic lethal is a term describes two mutants that individual is not lethal but together are lethal to cells. It not only reveals physical interactions of the gene products of synthetic lethal genes, but also reveals interactions along the same or parallel pathway. Methods to screen for genes that are synthetic lethal to each other are well established in yeast. However, it has not been established in mammalian culture cells. Since most of the cancer cells arise from mutations of normal cells, identification of genes that are synthetic lethal to genes mutated in cancer cells should enable the design of drugs that cause selective killing of cancer cells. Here we describe our attempt to establish a synthetic lethal screening system in cultured human cells. A cancer human cell line, H1299, which is deficient in PTEN was employed in our system. PTEN gene (phosphatase and tensin homologue located on chromosome 10) has been identified as a tumor suppressor gene. And it has been reported that the PTEN gene is mutated or deleted in several cancer cell types and tumor cell lines. The PTEN defect in H1299 cells was complemented by an episomal plasmid carrying wild type PTEN cDNA and an enhanced green fluorescent protein (EGFP) gene. The presence of this episomal plasmid could be monitored by EGFP. Mutation of genes that are synthetic lethal to PTEN should prevent the loss of EGFP fluorescence in H1299 cells. This established system will be used to screen for genes that are synthetic lethal to PTEN. Moreover, we also attemped to identify TEP1 synthetic lethal genes in yeast. In yeast, TEP1 is the homolog of the human tumor suppressor gene PTEN. TEP1 sequence has 28% identity and 45% similarity to human PTEN. Combining these two approaches should enable the identification of genes that are synthetic lethal to PTEN. More significantly, the identified genes will then be applied as a target for anti-cancer drug development.
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