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研究生:陳育侯
研究生(外文):Chen Yu-Hou
論文名稱:人類胎盤型及生殖細胞型鹼性磷酯基因於細胞中對糖皮質固醇差異表現之研究
論文名稱(外文):Differential Response of the Human Placental and Germ Cell Alkaline Phosphatases to Glucocorticoid in a Human Gastric Cancer Cell Line
指導教授:張自忠
指導教授(外文):Chang TC.
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:60
中文關鍵詞:鹼性磷酯糖皮質固醇
外文關鍵詞:Alkaline PhosphatasesGlucocorticoid
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人類熱穩定鹼性磷酯包含兩個接近相關基因所轉錄的同功:胎盤型鹼性磷酯,特別存在於胎盤組織;生殖細胞型鹼性磷酯主要表現於生殖型細胞中。在人類TMK-1胃癌細胞株中,糖皮質固醇會誘發胎盤型鹼性磷酯mRNA表現,丁酸鈉會誘發生殖細胞型鹼性磷酯mRNA表現。本文將胎盤型及生殖細胞型鹼性磷酯啟動區轉殖到螢光表現載體,轉染至TMK-1細胞中,以糖皮質固醇及丁酸鈉處理下,研究其藥物調控之差異。結果顯示,糖皮質固醇Dexmethasone會誘發胎盤型鹼性磷酯啟動區活性約10倍,雖然兩者啟動區僅有幾個核酸序列差異,但生殖細胞型鹼性磷酯啟動區受糖皮質固醇Dexmethasone遠較胎盤型低。我們確認受糖皮質固醇Dexmethasone調控之胎盤型鹼性磷酯啟動區為-296至-375,其中並沒有典型的糖皮質固醇反應序列。進一步將-375/-296此一片段與GREs和NF-1s比對,初步認為在-331/-296中包含非完美糖皮質固醇反應和類似NF-1序列。在抑制實驗中,我們使用糖皮質固醇接受子抗詰劑RU 486、蛋白質激C抑制劑H7及蛋白質激A抑制劑HA1004,結果顯示,RU 486會抑制80% Dexmethasone誘發胎盤型鹼性磷酯啟動區活性,H7會抑制40% Dexmethasone誘發胎盤型鹼性磷酯啟動區活性。丁酸鈉誘發生殖細胞型鹼性磷酯啟動區活性可被H7及HA1004抑制,不被RU 486抑制。在人類TMK-1胃癌細胞株中,Dexmethasone誘發胎盤型鹼性磷酯啟動區是經由糖皮質固醇接受子及蛋白質激C兩條途徑。

Human heat stable alkaline phosphatases are encoded by two closely related genes: the PAP, which specifies the term placental enzyme, and the GCAP, which is expressed primarily in germ cells. In the human gastric cancer cell line TMK-1 cells, glucocorticoid induced the accumulation of the mRNA of PAP, while sodium butyrate induced the expression of GCAP transcripts only. In order to study the mechanism underlying the differential regulation by glucocorticoid and sodium butyrate, PAP and GCAP promoters were fused to luciferase gene and trans fection were carried out in TMK-1 cells. Dexamethasone confers about 10 fold of induction in luciferase activity in PAP promoter. Despite that the PAP and GCAP promoters differ only by few bases, the GCAP promoter directs a very low level of response to dexamethasone. The dexamethasone response region in PAP promoter was identified between -296 to -375, though there is no discernible canonical glucocorticoid response element in this region. Detailed studies in the this region , demonstrated that the dexamethasone response region in placental alkaline phosphatase (PAP) promoter was identified between -296 to -331 which contains an NF-like and an imperfect glucocorticoid response element (GRE). Inhibition studies, using the glucocorticoid receptor antagonist RU486, protein kinase C inhibitor H7, and protein kinase A inhibitor HA1004, demonstrated that RU 486 blocked about 80% and H7 blocked about 40% of dexamethasone-mediated induction of PAP promoter activity. The sodium butyrate-induced GCAP activity can be blocked by H7 and HA1004 but not by RU486. These results suggest that in TMK-1 cells, the dexamethasone exerts its inductive effect on PAP through both of glucocorticoid receptor and the protein kinase C pathways.

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