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研究生:陳瑩真
研究生(外文):Yin-Chen Chen
論文名稱:突變性TSG101致癌能力的分析
論文名稱(外文):Analysis of oncogenic potential of mutated TSG101
指導教授:許金玉許金玉引用關係
指導教授(外文):Jin-Yuh Shew
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
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:62
中文關鍵詞:致癌能力乳癌細胞癌化程度
外文關鍵詞:breast canceroncogenic potentialtumorigenicityTSG101
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Breast cancer is the second leading cancer in Taiwanese women and the fourth leading cause of cancer mortality, which strongly indicate that it is an issue of particular public health importance. Its incidence is rapidly increasing in the past decade both in Taiwan and in other areas of Asia. The age of onset is much younger than their counterpart in western countries .The etiology of this disease remains elusive and preventable causes remain to be identified. At the molecular level, several oncogenes and tumor suppressor genes were shown to be involved in the development of breast cancer.
TSG101 was identified as a tumor susceptibility gene by homozygous functional inactivation of allelic loci in mouse 3T3 fibroblasts. Truncated Tsg101 transcripts, which are observed in a variety of human tumors as well as in small portion of the normal counterpart, have been attributed to aberrant RNA splicing and have been correlated with both cellular stress and mutation of p53. In our study, truncated transcripts could be found in 18.2% of normal and 56.9% of tumor specimens of breast cancer. Among them, the 247bp and 833bp truncated transcripts(termed TSG101-247 and TSG101-833, respectively) are found to be associated with systemic recurrence. However, the significance of these TSG101 splice forms in tumorigenesis remains elusive.
We generated several breast cancer cell lines (HBL-100 and MCF-7) stably expressing the TSG101-247 mutant or 833 mutant, and then evaluated the oncogenic potential of them by using tumorigenecity assays. From the soft agar colony formation experiments, it was apparent that transfection with TSG101-833 mutant increased the capacity of these cells to establish colonies to a highly significant degree. Therefore, We suggested that TSG101-833 mutant may be involved in the regulation of anchorage-independent growth of cells. However, there are no measurable differences in the TSG101-247 mutant transfected cells.
乳癌的發生對台灣地區的婦女而言,一直是十分重要的公共衛生問題,因為就婦女好發的癌症來看,乳癌在近年來一直是僅次於子宮頸癌排名第二位的癌症,另一方面,就婦女癌症的死亡率而言,也是排名第四位的女性癌症。更令人值得加以特別注意的是,台灣地區婦女乳癌發生率及死亡率在近二十年來急速上升,並且有越來越年輕化的趨勢。釵h研究致力於找尋與乳癌有關的危險因子,以企圖了解乳癌致病原因,希望能降低高發生率以及增加治療的效果。目前提出引發乳癌的危險因子有很多,就分子層次而言,一些致癌基因與抑癌基因的變異在乳癌檢體中出現,而這被認為與乳癌的形成具有相關性。
TSG101基因是經由將老鼠纖維母細胞(fibroblast)的基因進行隨機的踢除(random homozygous functional knockout),導致某些細胞細胞轉型,進而篩選到的具有抑癌潛能的基因。有些研究報告發現,在一些癌症中可偵測到異常(aberrant)的TSG101 transcripts,除了在癌組織中可偵測到,在少數鄰近的正常組織中也會出現,肇因為aberrant 或alternative splicing。有些研究指出,異常TSG101 transcripts會隨著逆境( cellular stress)、抑癌基因p53突變或tumor grade上升而出現越多。本實驗室分析乳癌病人檢體,亦偵測到一些異常TSG101 transcripts,進一步比較癌組織及鄰近正常組織,發現這些異常的transcripts大部分存在於癌組織中(56.9%),只有少部分鄰近正常組織會出現異常transcripts(18.2%);追蹤病人手術癒後狀況,發現若當初病人癌組織中同時存在TSG101-247及TSG101-833這兩種形式的異常transcripts,有相當高比例的病人會有手術後其他器官轉移復發(systemic recurrence)的情形。然而,TSG101-247 及TSG101-833這兩種形式的異常TSG101 transcripts,在細胞癌化的過程中扮演的角色,需要進一步的研究探討。
因此,我們建立了可以穩定並持續表達TSG101-247 mutant或TSG101-833 mutant的乳癌細胞株(HBL-100及MCF-7),進行細胞癌化程度分析,以了解這兩種形式的異常TSG101 transcripts是否具有致癌能力。實驗結果指出,在soft agar細胞群落生成實驗中,可穩定表現TSG101-833 mutant的細胞株,形成群落的能力會提升。我們推論,TSG101-833 mutant可能參與了細胞anchorage-independent growth的調控。然而,在我們所進行的細胞癌化程度分析實驗中,TSG101-247 mutant對於細胞癌化的影響並不顯著。
目 錄
頁數
中文摘要 1
英文摘要 3
第一章 緒論
第一節 文獻回顧 5
第二節 研究動機與實驗目的 9
第二章 材料與方法
第一節 實驗材料 12
第二節 細胞培養 13
第三節 突變性TSG101表達載體的構築 14
第四節 建立穩定表達突變性TSG101的細胞株 19
第五節 細胞癌化程度分析 22
第三章 實驗結果
第一節 突變性TSG101表達載體的構築 26
第二節 建立穩定表達突變性TSG101的細胞株 26
第三節 細胞癌化程度分析 27
第四章 討論 32
圖表
圖一 TSG101-247 mutant 及TSG101-833 mutant
轉譯區示意圖 35
圖二 實驗過程中所用到的載體 36
圖三 TSG101-833 mutant 表達載體構築的流程 37
圖四 限制酶圖譜分析選殖到pRc/CMV載體上的
TSG101-833 mutant DNA片段 39
圖五 TSG101-247mutant 表達載體構築的流程 40
圖六 限制酶圖譜分析選殖到pcDNA3.1載體上的
TSG101-247 mutant DNA片段 42
圖七 能穩定表現TSG101-833 mutant的細胞株之選 43
圖八 能穩定表現TSG101-247 mutant的細胞株之篩選 44
圖九 能穩定表現TSG101-833 mutant的HBL-100細胞株
的癌化程度分析 46
圖十 能穩定表現TSG101-833 mutant的MCF-7細胞株
的癌化程度分析 48
圖十一 能穩定表現TSG101-247 mutant的MCF-7細胞株
的癌化程度分析 50
圖十二 能穩定表現TSG101-247 mutant的HBL-100細胞株
的癌化程度分析 52
表一 本實驗所使用細胞株的背景 54
表二 TSG101 primer的list及PCR反應的條件 55
表三 TSG101-833 mutant影響細胞癌化的總整理 56
表四 TSG101-247 mutant影響細胞癌化的總整理 57
參考文獻 59
馬家琳 (1998) 台灣地區肺癌檢體中TSG101基因之研究。台灣大學醫學院生物化學暨分子生物學研究所碩士論文

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