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研究生:李弘揚
論文名稱:甲狀腺素受體對於運鐵蛋白的調控
論文名稱(外文):Regulation of transferrin gene by thyroid hormone receptors
指導教授:林光輝林光輝引用關係
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:2001
畢業學年度:89
語文別:中文
論文頁數:73
中文關鍵詞:甲狀腺素甲狀腺素受體運鐵蛋白調控
外文關鍵詞:thyroid hormonethyroid hormone receptorstransferrinregulation
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摘要
運鐵蛋白 (transferrin)屬於一種醣蛋白,其主要功能為攜帶血液中游離的鐵離子。而運鐵蛋白主要在肝臟內製造,而後被釋放到血液中。所以運鐵蛋白所扮演的角色為提供細胞生長所需的含鐵量與維持血液中鐵離子的恆定。T3為有活性的甲狀腺素,影響細胞的生長,代謝,分化;甲狀腺受體(TR)存在於細胞核內,為一個甲狀腺素依賴型的轉錄因子,藉由與 T3的作用,再一起結合到甲狀腺素位元(thyroid response element,TRE)上,調控特定基因的表現。
以人類肝癌細胞株 Hep-G2為實驗材料,使用stable transfection的方法,使其表現高量的 TRa 1蛋白;使用transiet transfection的方法,確定了TR的transactivation的能力。另外,給予Hep-G2 stable clone細胞100nM T3的刺激,會觀察到細胞的生長有被壓抑的現象。使用Western blot及Northern blot分析Hep-G2 stable clone細胞的運鐵蛋白,發現在不同時間及不同濃度的 T3處理後,運鐵蛋白都有增加的趨勢,顯示運鐵蛋白會受到 T3的調控。
總結來說,T3藉由高表現的 TR影響細胞的生長及運鐵蛋白基因與蛋白的活化。

Abstract
Transferrin is an iron binding protein expressed in all mammals, and mainly synthesized in the liver and secreted into the bloodstream. Therefore, transferrin appears to play an important role in local iron homeostasis for organism survival and growth. T3 is an active form of thyroid hormone, affecting cell growth, differentiation and metabolism. Thyroid hormone receptors (TRs) is an nuclear protein and a ligand dependent transcription factor, which function by binding to the specific thyroid hormone response sequence (TRE) to regulating transcription of target genes.
In this study, we utilized stable transfection method on Hep-G2 cell line, as a result, these Hep-G2 stable clones highly expressed TRa1 gene. Then, we confirm the transactivation activity by transient transfection a luciferase plasmid containing a positive TRE sequence, LAP. In addition, Hep-G2 cells have a suppressed growth rate when treated with high dose T3. And then, in Western and Northern blotting result, we found that transferrin is increased in protein and mRNA levels, both in a time dependent manner and a dose dependent manner when treated with T3. It reveals that transferrin is regulated by T3.
In summary, T3 can regulate the expression of transferrin gene and cell growth through TRs.

第一章 緒論1
第一節 鐵離子的重要性1
第二節 運鐵蛋白 (Transferrin)4
第三節 甲狀腺素 (Thyroid Hormone)11
第四節 甲狀腺素受體 (Thyroid Hormone Receptors)12
第五節 甲狀腺荷爾蒙反應位元 (Thyroid Hormone Response Element, TRE)18
第二章 材料與方法22
定量蛋白質 (protein quantifacation)22
蛋白質的萃取 (protein isolation)22
西方墨點 (Western Blotting)23
T3的配製(T3 preparation)25
T3 depleted (Td) serum 的配製26
培養細胞 (cell culture)26
轉殖 (transfection)27
Luciferase assay28
Hep-G2 Wt細胞株的製備29
Tf cDNA plasmid 的製備30
北方墨點 (Northern Blotting)34
第三章 結果38
G2 stable clone 表現大量 wildtype-TR1蛋白38
TR1 在細胞內具有 transactivation的功能39
T3對 stable clone Hep-G2細胞的生長造成壓抑40
plasma蛋白的篩選40
T3對於 transferrin在蛋白質上的調控41
T3對於 transferrin在mRNA上的調控43
第四章 討論45
第五章 參考文獻50

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