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博碩士論文 etd-0828101-111807 詳細資訊
Title page for etd-0828101-111807
論文名稱
Title
甲狀腺腫瘤檢體中TSG101與RET基因表現之研究
The expression of TSG101 and RET gene in thyroid carcinoma specimens.
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
65
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2001-07-25
繳交日期
Date of Submission
2001-08-28
關鍵字
Keywords
免疫組織染色、甲狀腺腫瘤
TSG101, immunohistochemistry, RET, RT-PCR
統計
Statistics
本論文已被瀏覽 5684 次,被下載 3608
The thesis/dissertation has been browsed 5684 times, has been downloaded 3608 times.
中文摘要
本論文之主要目的為分析臨床甲狀腺檢體中腫瘤易感基因TSG101 及致癌基因RET的表現情況。
TSG101是一腫瘤易感基因,當其功能失去活性後會導致老鼠纖維母細胞發生轉形 (transformation)的現象,且此轉形細胞在裸鼠體內會產生轉移性的腫瘤,顯示TSG101具有腫瘤抑制基因之特性。許多針對癌組織genomic DNA之分析顯示,此基因在癌組織中並無缺失現象,因此TSG101並非典型之腫瘤抑制基因。許多後續報告指出TSG101在癌組織中,常有RNA剪接異常現象,與細胞緊迫及p53狀況有相關性,這些異常TSG101 RNA之出現,雖然可能與癌症的成因無關,但可反應癌症形成的細胞內環境。有趣的是,最近的研究發現,TSG101參與與癌症形成息息相關的MDM2/p53回饋控制。基於上述之發現,我們以TSG101多株抗體進行了免疫化學組織染色,研究各類型甲狀腺癌之TSG101表現的情況,以探討TSG101蛋白表現與甲狀腺癌間之相關性,檢體包含了48例乳突性甲狀腺癌、21例濾泡性癌、7例髓質性癌及5例未分化性癌。結果顯示乳突性癌有100%、濾泡性癌有85%、髓質性癌有91% 而未分化性癌則有60% 的檢體具有第3等級染色以上之陽性反應,而其周圍之正常濾泡組織則呈現陰性或局部點狀細胞質染色且偶而在核內可見染色,顯示在甲狀腺腫瘤中,普遍有TSG101蛋白過度表現的現象。在乳突性癌檢體中確定有TSG101過度表現現象,而17個檢體經steadiness box區域cDNA定序分析,結果顯示並無基因變異現象。因此TSG101的過度表現並不是因為此區域突變所造成,而可能與RNA穩定度或promoter的活性有關。本研究結果顯示TSG101的表現與人類甲狀腺癌的腫瘤生成之相關性,值得進一步加以探討。


在各種甲狀腺腫瘤中ret原致癌基因藉由染色體重組而活化的情形,只發生在甲狀腺乳突性癌中,其所形成的重組基因稱為ret/PTC。乳突性癌的發生與環境的因素有關,在非放射線污染之區域其ret/PTC的盛行率為0-55%。為了要了解台灣的ret/PTC表現情形為何及找尋新型ret/PTC,我們採Multiplex PCR方法,從62例乳突性癌甲狀腺檢體中篩選出具有ret/PTC基因重組的檢體,並以類型特異性引子進行PCR放大相關之ret/PTC基因,並經定序分析加以確認。結果顯示,62例的檢體中,ret/PTC1及ret/PTC3各有兩例,另有一例為ELKS-RET。從Multiplex PCR中,有17個檢體可能有新的ret基因重組型,將可進一步利用5’RACE實驗來加以確認。
Abstract
The aim of this thesis is to evaluate the expression of both TSG101 tumor susceptibility gene and ret oncogene in human thyroid carcinoma specimens.
Functional inactivation of TSG101 in mouse fibroblast leads to cellular transformation and the ability to form metastatic tumors in nude mice. No genomic deletion of TSG101 gene has been reported in human cancer, casting a doubt on the role of TSG101 as a classical tumor suppressor. Subsequent studies reveal that TSG101 is a frequent target of spilicing defects, which is correlated with cellular stress and p53 status, and might reflect the cellular environment during the cancer development. Furthermore, recent reports demonstrate TSG101 as a part of the MDM2/p53 regulatory circuitry, a well recognized circuitry that upon deregulation results in tumorigenesis. In this study we have analyzed TSG101 gene expression in 85 specimens of thyroid carcinomas. The results indicated that 100% of papillary carcinomas (48/48), 85% of follicular carcinomas (18/21), 91% of medullary carcinomas (10/11) and 60% of undifferentiated carcinomas (3/5) showed strong to moderate cytoplasmic staining, whereas the staining was completely negative, or cytoplasmic dot-staining in the adjacent non-neoplastic follicular cells. Occasionally, the staining could be found in the nucleus. Subsequently, sequence analysis of 17 papillary carcinoma specimens revealed no mutation in steadiness box region, indicating that it might not be the cause of TSG101 protein overexpression. In summary, our results indicate strong correlation of TSG101 overexpression and thyroid carcinomas. Further experiments are urged to clarify the relationship of TSG101 overexpression and thyroid tumorigenesis.
Rearrangement of ret proto-oncogene is unique to papillary thyroid carcinoma (PTC). These rearrangements consist of the fusion of ret tyrosine kinase domain to a variety of heterologous genes, thus generating chimeric transforming oncogenes termed, ret/PTC. The frequency of ret/PTC activation in non-radiation exposured adult populations has been reported to vary from 0-55% depending on the geographic distribution. To detect ret rearrangement and to identify candidate of novel ret/PTC in 62 specimens of PTC collected from southern Taiwan, a RT-multiplex PCR method was used to reveal the possible specimens that harbor ret rearrangements. Type specific-PCR amplification and subsequent sequence analysis of PCR product were performed to identify the known types of ret/PTC. We have identified two cases of ret/PTC1, two cases of ret/PTC3 and one case of ELKS-RET. Excitingly, four cases of unknown ret/PTC type were identified. Hence, 5’-RACE strategy will be employed to identify novel ret/PTC in these four specimens.
目次 Table of Contents
中文摘要……………………………………………………2
英文摘要……………………………………………………4
背景介紹 …………………………………………………12
材料與方法 ………………………………………………27
結果與討論 ………………………………………………38
參考資料 …………………………………………………43
圖表 ………………………………………………………65
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