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研究生:唐凱倫
研究生(外文):Kai-Lun Tang
論文名稱:探討蛋白精胺酸甲基轉移酶1(PRMT1)對K562細胞分化及基因表現的影響
論文名稱(外文):Study on the effect of PRMT1 on K562 differentiation and gene expression.
指導教授:林蔚靖
指導教授(外文):Wey-Jinq Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:59
中文關鍵詞:蛋白質精胺酸甲基化
外文關鍵詞:Protein arginine methylation
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PRMT 1 為哺乳動物中主要的蛋白質甲基轉移酶,負責精胺酸的甲基化,目前許多研究顯示 PRMT 1 能參與在調控細胞生長、基因表現以及訊息傳導等。實驗室先前發現穩定過量表現 PRMT 1 會抑制 PMA 誘導慢性骨髓白血病細胞株 K562 走向巨核細胞系分化,目前廣泛使用的蛋白傳導系統其中一類為 TAT 系統,與外來蛋白以形成融合蛋白的方式能快速並且高效率的送入細胞。
本研究嘗試將 PRMT 1 以 TAT 蛋白傳導方式送入 K562 細胞中,觀察其進入細胞及對巨核系分化的影響,以西方墨點法觀察到重組 TAT-PRMT 1 能快速進入細胞。而藉由細胞形態改變及特定細胞表面標記 CD 41 的表現能判定 TAT-PRMT 1 抑制 PMA 誘導的巨核系分化,其抑制效果在 PMA 誘導後兩小時加入 TAT-PRMT 1 的情況下具有最顯著變化,顯示出具抑制作用的受質甲基化並不會發生於 PMA 誘導之前。另一方面部分轉錄因子如: GATA-1 、 GATA-2 及 Runx 1 在巨核系分化中扮演重要角色,於本研究我們利用定量 PCR (real-time PCR) 發現異常表現 PRMT 1 會抑制轉錄因子表現量,由結果顯示 PRMT 1 可能藉由負調控與分化相關的基因而達到抑制 PMA 所誘導分化的結果。
Protein arginine methyltransferase 1 (PRMT1), the predominant protein arginine methyltransferase in mammalian cells, is responsible for arginine methylation and is involved in development, gene expression and signal transduction. In this study, I introduced PRMT1 into human leukemia K562 cells by a TAT-mediated protein transduction system. The purified TAT-PRMT1 entered cells rapidly as examined by Western blotting. TAT-PRMT1 suppressed PMA-induced megakaryocytic differentiation as assessed by cytological changes and expression of the specific surface marker CD41. Suppression was most profound when TAT-PRMT1 was introduced two hours after PMA treatment, indicating that substrate methylation responsible for the suppressive effect did not have to occur before PMA treatment. Transcription factors such as GATA-1, GATA-2, and Runx 1 have been shown to play critical roles in megakaryocytic differentiation. In this study, we showed that ectopic expression of PRMT1 suppressed expression of these transcription factors as examined by real-time RT-PCR. These results indicate that PRMT1 negatively regulates expression of differentiation-specific genes and may thus suppress
megakaryocytic differentiation induced by PMA.
目 錄 …………………………………………………………………………… 1
縮 寫 表 …………………………………………………………………………… 2
圖次目錄 …………………………………………………………………………… 4
中文摘要 …………………………………………………………………………… 5
英文摘要 …………………………………………………………………………… 6
緒 論 …………………………………………………………………………… 7
研究目標 ……………………………………………………………………………15
實驗材料 ……………………………………………………………………………16
實驗方法 ……………………………………………………………………………20
實驗結果 ……………………………………………………………………………27
討 論 ……………………………………………………………………………33
參考文獻 ……………………………………………………………………………37
圖 表 ……………………………………………………………………………41
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柳鈞翔, TPA刺激K-562細胞分化過程中蛋白質甲基化之研究 國立陽明大學生物藥學所 碩士論文 2004
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