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系統識別號 U0026-2507201118030600
論文名稱(中文) 利用人類血癌細胞模型探討 cAMP 對於佛波脂誘導巨核細胞分化的調控
論文名稱(英文) A Study of cAMP Action on Phorbol Ester-induced Megakaryocyte Differentiation in Human Erythroleukemia Cell Lines
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 99
學期 2
出版年 100
研究生(中文) 林育年
研究生(英文) Yu-Nian Lin
學號 s26981107
學位類別 碩士
語文別 中文
論文頁數 72頁
口試委員 口試委員-陳清玉
口試委員-錢偉鈞
指導教授-簡偉明
中文關鍵字 巨核細胞  分化  環腺苷酸單磷酸 
英文關鍵字 Megakaryocyte  differentiation  cAMP 
學科別分類
中文摘要 巨核細胞由骨髓中的造血幹細胞分化而來,後生成血小板。已知人類血癌細胞株 HEL和K562 在PMA誘導下,具有細胞變大、細胞表面抗原 CD41/61 表達增加和染色體套數增加等巨核細胞分化時的特性。且過去研究指出,在巨核細胞分化中,胞外訊息調節激酶 (Extracellular-signal-regulated kinase,ERK) 持續地活化 (>24h) 扮演相當重要的角色。先前實驗室發現,cAMP 會阻止巨核細胞分化,因此為了更深入探討 cAMP 阻止巨核細胞分化的機制,我們利用 HEL 和 K562 細胞研究在腺苷酸環化酶 (Adenylyl cyclase) 活化劑 Forskolin (FSK) 或 cAMP 類似物: dibutyryl- cAMP (dbcAMP) 前處理下,觀察巨核細胞分化的情形。流式細胞儀分析的結果顯示: FSK 抑制了 PMA 誘導巨核細胞分化時的特性,包括細胞變大、染色體套數增加和細胞表面抗原 CD61 的表達。而西方墨點法分析的結果顯示:FSK 除了抑制 ERK 磷酸化和增加 PKC α/βII 磷酸化之外,還會使原本 PMA 誘導的細胞週期相關之蛋白: Retinoblastoma protein (Rb) 和 Cyclin- dependent kinase 4 (CDK4) 表現量下降。進一步利用 EPAC 致效劑 (8-pCPT-2’-O-Me-cAMP) 和 PKA 致效劑 (N6- benzoyl-cAMP) 探討 cAMP 是經由哪一個下游訊息傳遞路徑影響巨核細胞分化,結果發現 PKA 致效劑前處理的 HEL 細胞,其細胞分化時的形態、細胞大小、染色體套數和表面抗原 CD61 皆會受到抑制。此外為了探討 cAMP 導致 Rb 表現量下降的機制,我們對 HEL 細胞投予 PKA 抑制劑,證實 cAMP 也是透過 PKA 來使 Rb 的蛋白表現量下降。綜合以上的實驗結果我們歸納出, cAMP 是透過活化 PKA 來抑制巨核細胞分化的特性和 Rb 蛋白表現量的增加,進而阻止巨核細胞分化。
英文摘要 Megakaryocytes (MKs), the bone marrow cells for platelet production, are derived from hematopoietic stem cells (HSCs). HEL and K562 cells have the typical properties of megakaryocytic progenitors and have the abilities to differentiate into MKs in response to phorbol 12-myristate 13-acetate (PMA). Previous studies have shown that sustained ERK activation (>24h) is required for MK differentiation. Our data showed that cAMP reduced PMA-induced cell size enlargement, DNA ploidy, cell surface marker, CD61, expression. Therefore, we investigated the pathway involved in cAMP action by adenylyl cyclase activator forskolin (FSK) or cAMP analog dibutyryl-cAMP (dbcAMP) pretreatment of HEL and K562 cells. The data shown that increase in intracellular cAMP attenuates PMA-mediated MK differentiation, which involved down-regulation of ERK1/2 phosphorylation and up-regulation of PKCα/βII phosphorylation. And the expression of cell cycle-related proteins, retinoblastoma protein (Rb) and cyclin-dependent kinase 4 (CDK4), which is upregulated by PMA, were decreased either. Futhermore, PMA-induced cell size enlargement, DNA ploidy, cell surface marker, CD61, expression were attenuated by PKA activator, which is one of cAMP downstream signaling. Conversely, inhibition of PKA recovered the expression of Rb. These results indicate that cAMP, via PKA activation, and its downstream signaling attenuates PMA-induced MK differentiation, which is through Rb down-regulation.
論文目次 口試合格證明 I
摘要 II
Abstract III
誌謝 IV
目錄 V
圖目錄 VI
縮寫表 VIII


緒論 1
材料與方法 10
實驗結果 21
討論 29
結論 35
參考文獻 36
附錄 46
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