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研究生:吳介凱
研究生(外文):Jie Kai Wu
論文名稱:鑑定咽喉癌中之抗放射線基因
論文名稱(外文):Identification of the radioresistant genes in pharyngolaryngeal caner
指導教授:鄭恩加
指導教授(外文):A. J. Cheng
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:62
中文關鍵詞:咽喉癌放射線抗性
外文關鍵詞:pharyngolaryngeal cancerradioresistance
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在頭頸癌中,由於咽喉控制聲帶以及吞嚥等重要生理功能,所以咽喉癌的發生對病患有著非常嚴重的影響。為了要控制腫瘤且達到器官保存,在咽喉癌的治療策略往往使用放療結合化療來達到器官保存的目的。然而,非所有患者對放療都有良好反應,因此造成放療治療失敗。所以確定分子層面對於放療的影響,進而幫助預測病患個體對於放射線的敏感度及使放療效率增加到最大,是目前非常急迫的課題。
為了確定放射線抗性相關基因,我們在兩株咽喉癌細胞株使用了分次化放射線療法來建立放射線抗性子代細胞株,每次使用2 Gy累積至60 Gy以上。Detroit抗放射線子代細胞株比起Detroit母代細胞株更具有放射線抗性,但在老化或是細胞增生上沒有很明顯的變化。FaDu抗放射線子代細胞株生長比母代細胞快速,但放射線抗性及老化現象卻沒有明顯改變。我們進一步使用cDNA microarray來篩選在Detroit母代及抗放射線子代細胞株中表現有差異的基因。在Detroit抗放射線子代細胞株中,共有18個及17個基因分別被兩倍以上高度及低度表現,這些基因可能與抗放射線有關;並使用array comparative genome hybridization 來比較FaDu母代與抗放射線子代細胞在基因體層級上放射線所造成的不正常現象,在FaDu抗放射線子代細胞株上發現染色體2、4及11不正常缺失,染色體7、9、17和20上有不正常增生。
另一方面,之前實驗室從口腔癌抗放射線子代細胞株篩選出的10個蛋白,在咽喉癌中進行更多抗放射線功能鑑定。Annexin II與細胞骨骼有關,在咽喉癌之抗放射線子代細胞株都都有被顯著高度表現,annexin II表現可能有助於放射線抗性。為了解annexin II與放射線的關係,將能抑制annexin II的siRNA建構至質體載體內,並轉染入咽喉癌細胞探討對於放射線敏感度可能的影響。抑制annexin II表現量對於放射線敏感度在細胞及生化階層都已經被探討。實驗結果顯示annexin II可能被放射線敏感的誘導,NF-B則是永久性的增加表現量。在24對頭頸癌臨床檢體中發現,annexin II被過量的表現接近總體的75%。
In head neck cancer, pharyngolaryngeal cancer (PLC) is a very critical disease because the anatomy site controls vocal and swelling function. In order to achieve both tumor control and organ preservation, the policy of treatment modality of PLC tends to apply primary radiotherapy and combined with chemotherapy for organ preservation. However, not all patients response to radiotherapy causing treatment failure, it is an urgent need to determine molecule signatures that can predict radiosensitivity to maximum the therapeutic efficacy individually.
To determine radioresistant associated genes, we have established two radioresistant (RR) sublines of PLC cancer cell lines, FaDu and Detroit, by fractionated irradiation of 2Gy to a total dose of 60 Gy. The Detroit-RR sublines were more radioresistant than parental, but had no significant difference on proliferation and senescence. The FaDu-RR grew with faster rate than parental, but did not have significant differences on radioresistance and senescence. The differential gene expression profile between Detroit parental and RR cells has been determined using cDNA microarray method. Total of 18 genes were found 2-fold of over-expression and 17 were found down-regulated in the Detroit-RR cells, indicating potential association of these genes in RR phenotypes. Array comparative genome hybridization (ArrayCGH) was used to detect the abnormal amplification or deletion of FaDu-RR sublines. FaDu-RR had deletion on chromosome 2, 4, and 11 and amplification of on chromosome 7, 9, 17, and 20.
On the other hand, 10 proteins that were previously screened in the RR sublines of oral caner cells were further investigated for their potential functions in PLC cells. Among which, a cytoskeleton associated gene annexin II, were found significant over-expressed in two PLC-RR subline cells, suggesting this gene may contribute to radioresistance. To investigate its function, siRNA plasmid against annexin II was cloned and trasnfected to PLC cells to examine the potential alteration in radiosensitivity. Cellular and biochemical studies of radiosensitivity after annexin II knockdown were proceed. The outcome indicated that annexin II might induced by radiation sensitively, and NF-B permanently upregulated in RR sublines. In head and neck cancer clinical samples, we also found that Annexin 2 overexpressed approximately 75% in 24 patients.
Background 1
Study Aim and Research Design 9
Material and Method 11
Result 19
Discussion 27
REFERENCES 30
Appendix 37
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