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研究生:葉明泓
研究生(外文):Ming-Hong Yeh
論文名稱:甲狀腺素對PCTAIRE-1之正向調控
論文名稱(外文):The expression of PCTAIRE-1 is positively regulated by thyroid hormone
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
外文關鍵詞:PCTAIRE-1Thyroid hormone
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甲狀腺素在細胞代謝、分化以及發育方面扮演著一重要角色。在甲狀腺素受體(TRα)過度表現之肝癌細胞株(HepG2)中,利用cDNA microarrays技術分析受到甲狀腺素調控之基因,其中包含許多kinases,PCTAIRE-1即為其一。
在甲狀腺素受體過度表現之肝癌細胞株中,於甲狀腺素刺激48小時後,PCTAIRE-1蛋白質表現量約有7.59倍的上升,PCTAIRE-1 mRNA表現量約有3.86倍的上升。一種蛋白質轉譯抑制劑(cycloheximide)可抑制甲狀腺素對於PCTAIRE-1 mRNA的正向調控,證明此種調控為間接地。此種調控也發現於其他過度表現甲狀腺素受體之肝癌細胞株,此外,類似的結果也在動物模式中被證實。為了確知是何區域受到甲狀腺素之間接調控,故建構出具有不同PCTAIRE-1啟動子區域之活性測試載體,發現甲狀腺素會促使啟動子活性大約1.65~2.25倍的上升,至於是何種轉錄因子調控PCTAIRE-1之表現,還需更進一步的研究。綜合以上所述,甲狀腺素對於PCTAIRE-1之調控為正向間接地。最後我們利用RNAi方式降低PCTAIRE-1之表現,進而測試對於細胞增殖之影響。未來之研究可朝甲狀腺素如何藉由調控PCTAIRE-1來影響生理機能方面探討。
Thyroid hormone, 3, 3’, 5-triiodo-L-thyronine (T3) regulates cell metabolism, differentiation, and development. cDNA microarrays were performed to study the mechanism of target gene regulation after T3 treatment in a thyroid hormone receptor α (TRα)-over-expressing hepatoma cell line (HepG2-TR). The differentially expressed target genes are several kinases, including PCTAIRE-1.
PCTAIRE-1 was elevated roughly 7.59-fold in HepG2-TRa cells at the protein level after 48 hr of T3 treatment and 3.86-fold increase at the mRNA level after 48 hr T3 treatment. The protein synthesis inhibitor, cycloheximide, inhibits T3-induced PCTAIRE-1 expression, suggesting that regulation was indirect. Induced expression of PCTAIRE-1 was observed in other TR expressing HepG2 cells also. Similar findings were obtained from thyroidectomized rats after T3 application. To identify and localize the critical regulatory site, series of deletion mutants of PCTAIRE-1 promoter fragments in pGL3 plasmid were constructed. The promoter activity of the PCTAIRE-1 gene was induced roughly 1.65- to 2.25-fold by T3. Further study needs to find which transcription factor involving in this regulation. In summary, this study demonstrated that the PCTAIRE-1 gene was indirectly positively regulated by T3. Finally, we reduce PCTAIRE-1 expression by RNAi and elucidate its effect on cell proliferation. Further study is required to determine the physiological role of PCTAIRE-1 induction mediated by T3.
指導教授推薦書…………………………………………………………
口試委員會審定書………………………………………………………
授權書…………………………………………………………………...iii
誌謝……………………………………………………………………...iv
中文摘要…………………………………………………………………v
英文摘要………………………………………………………………...vi
目錄……………………………………………………………………..vii
圖表目錄………………………………………………………………...ix
緒論………...…………………………………………………………..1
甲狀腺素調控之介紹……………………………………………..1
甲狀腺素………………………………………………………..1
甲狀腺素受體…………………………………………………..2
甲狀腺素反應元………………………………………………..4
與甲狀腺素受體相互作用之調節因子………………………..4
PCTAIRE-1之背景與特性……………………………………….5
研究動機…………………………………………………………..7
材料與方法……………………………………………………………..8
細胞培養…………………………………………………………..8
3, 3’, 5-triiodo-L-thyronine (T3)溶液之配製…………….………8
T3-depleted serum (Td)之配製………………………………….8
RNA與蛋白質萃取前之細胞處理……………………………….9
RNA之萃取……………………………………………………….9
定量反轉錄聚合酶連鎖反應……………………………………..9
北方墨點法……………………………………………………....10
蛋白質之萃取…………………………………………………….11
蛋白質之定量…………………………………………………….11
西方墨點法……………………………………………………….11
動物實驗………………………………………………………….13
啟動子活性測試…………………………………………..……...13
PCTAIRE-1表現抑制之細胞株建構……………….………….14
細胞增殖實驗…………………………………………................14
統計方法……………………………………………….………...14
結果…………………………………………………..………………..15
甲狀腺素對PCTAIRE-1之正向調控 - mRNA level (I)……15
甲狀腺素對PCTAIRE-1之正向調控 - mRNA level (II)…..15
甲狀腺素對PCTAIRE-1之正向調控 - mRNA level (III)….15
甲狀腺素對PCTAIRE-1之正向調控 - Protein level............16
動物體內甲狀腺素對PCTAIRE-1之正向調控…………....….18
甲狀腺素對PCTAIRE-1之調控為間接地……………….…….18
甲狀腺素對PCTAIRE-1 promoter之正向間接調控區域…….19
正常肝細胞與腫瘤肝細胞間PCTAIRE-1表現之差異….20
PCTAIRE-1對細胞增殖之影響…………………………..20
結論………….………………………………………………......22
甲狀腺素與PCTAIRE-1之調控……………….………...22
甲狀腺素對PCTAIRE-1調控之分子機制……………….22
PCTAIRE-1功能之研究………………………………….23
參考文獻………………………………………………………….25
附錄………………………………………………………………..29
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