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研究生:張雅婷
研究生(外文):Ya-Ting Chang
論文名稱:甲狀腺素對stathmin逆向調控機制之研究
論文名稱(外文):The Mechanism of How Thyroid Hormones Negatively Regulate the Expression of Stathmin
指導教授:林光輝林光輝引用關係
指導教授(外文):Kwang-Huei Lin
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:65
中文關鍵詞:甲狀腺素
外文關鍵詞:thyroid hormonesstathmin
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甲狀腺素 (3, 3’, 5-triiodo-L-thyronine, T3)是一可調控細胞生長、發育與分化的重要因子,其受體 (Thyroid hormone receptor, TR)屬於固醇類荷爾蒙受體家族之一,是一甲狀腺素依賴型的轉錄因子,而甲狀腺素便是藉由細胞核內的甲狀腺素受體將其訊號傳至細胞內。我們利用cDNA microarray分析技術研究在過度表現甲狀腺素受體的肝癌細胞株 (HepG2-TRα)中,甲狀腺素對下游基因的調控,在受到甲狀腺素的負向調控的基因中,我們選擇受調控較明顯的基因進行研究,Stathmin是其中之ㄧ。Stathmin又名Op18 (oncoprotein 18),會調控microtubule stabillity進而影響cell proliferation、cell motility。以HepG2-TRα1 cell進行cDNA microarray分析發現,經T3處理48小時後stathmin表現量下降約3.5倍;而stathmin mRNA及protein level在經T3處理後均可見顯著下降的情形。加入轉譯抑制劑 (Cycloheximide)並不會影響細胞內T3對Stathmin之調控,顯示其調控機制為甲狀腺素與其受體直接結合在stathmin基因上游啟動子之逆向甲狀腺素位元 (Negative Thyroid hormone responsive element, nTRE)上,促使基因表現量下降;Promoter assay結果顯示stathmin promoter region的確受TR逆向調控。利用Retrovirus shRNA based RNAi技術建立stathmin表現持續被抑制的細胞株,與HepG2-TRα1比較細胞增生的情形,發現stathmin表現持續被抑制的兩株細胞株生長速度的確比HepG2-TRα1慢,且細胞的生長速度與細胞內stathmin的表現量成正相關。
Thyroid hormone (triiodothyronine, T3) regulates development, growth and metabolism in many organs and tissues. Most of the effects of thyroid hormone are mediated by the thyroid hormone receptors (TRs). TRs function as transcription factors to increase or decrease levels of gene expression. To study the downstream genes regulated by T3 in TR overexpressing HepG2 cell line, cDNA microarray was performed. Approximately 4 % (291 of 7600) genes were negatively regulated by T3, including stathmin (STMN1,Op18). Stathmin is an oncoprotein and a mitotic regulator that function via its ability to regulate microtubule dynamics. Stathmin modulates microtubule stability by promoting depolymerization of microtubule and/or preventing polymerization of tubulin heterodimers. Stathmin high expression is associated with cell proliferation and is reported in many kinds of cancer. However, the transcription mechanism or its physiological role responsible for this regulation remains poorly understood. In this study, we concentrated our efforts on investigating the regulation of stathmin due to T3 treatment. Stathmin transcription in HepG2-TR#1 cell lines was repressed ~35%, and ~36% at the protein level, 12hr or 36hr after T3 treatment. The protein synthesis inhibitor, cycloheximide, did not rescue the repression of stathmin by T3, indicating that this regulation was direct. To further localize the putative negative thyroid hormone response element (n-TRE), the stahmin promoter was cloned in pGL3 plasmid and activities were assayed. The activity of the region containing -2069/-1137 fragment was still repressed ~50% by T3. To further understand the consequence of stathmin repression, we knock-down the stathmin expression in HepG2-TR#1 by siRNA. In stathmin knock-down stable cells, cell proliferation was repressed. These results indicate that T3 might play an important role in the control of cell proliferation mediated by the stathmin.
中文摘要 i
英文摘要 iii
緒論 1
甲狀腺素(Thyroid hormone) 1
甲狀腺素(Thyroid hormone) 1
甲狀腺素受體(Thyroid hormone receptors) 2
甲狀腺素相關疾病 5
Stathmin及其作用機制 6
材料與方法 9
細胞培養(Cell culture) 9
Triiodo-thyronine(T3)的配製 9
T3 depleted (Td) serum的配製 9
RNA萃取(RNA extraction) 10
即時定量聚合酶連鎖反應(Real-time quantitative-PCR) 10
北方墨點法(Northern blot) 11
蛋白質萃取(Total protein extraction) 12
組織蛋白質萃取(Tissue protein extraction) 12
蛋白質定量(Protein quantification) 12
西方墨點法(Western blot) 13
動物模式的建立 14
質體建構(Plasmid construction) 14
轉殖(Transfection) 15
Luciferase分析法(Luciferase assay) 15
建立穩定抑制stathmin表現之細胞株(Establishment of stathmin knock-down stable cell lines) 16
細胞增生分析法(Cell proliferation assay) 16
細胞週期分析法(Cell cycle analysis) 17
結果分析(Data analysis) 17
結果 18
cDNA微陣列(cDNA micro-array)分析結果 18
即時定量聚合酶連鎖反應偵測T3處理後細胞內STMN1表現情形 18
北方墨點法偵測T3處理後細胞內STMN1表現情形 18
西方墨點法偵測T3處理後細胞內stathmin表現情形 19
西方墨點法偵測甲狀腺切除之大鼠(rat)肝組織中的表現量 20
西方墨點法偵測肝癌病人肝組織中stathmin的表現量 21
分析T3對stathmin之逆向調控機制是否為直接調 21
分析T3對stathmin基因上游啟動子之活性影響 22
HepG2-TR1#1的細胞增生受T3抑制之情形 22
HepG2-TR1#1的細胞週期受T3抑制之情形 23
西方墨點法偵測不同的肝癌細胞株中sathmin的表現量 24
西方墨點法偵測穩定抑制stathmin表現量之細胞株中stathmin的表現量 25
穩定抑制stathmin表現量之細胞株細胞增生的情形 25
比較HepG2-TR1#1與穩定抑制stathmin表現量之細胞株細胞週期的變化 25
討論 27
參考文獻 31
附錄 42
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