臺灣博碩士論文加值系統

Mitogen-activated protein kinase (MAPK) 訊息傳遞路徑廣泛調控許多細胞功能，例如細胞生長、凋亡及分化。文獻中，Erk MAPK活化促進巨核球細胞系分化，p38 MAPK則具負調控作用。K562細胞為人類血球系分化的前驅細胞，受到PMA (phorbol 12-myristate 13-acetate) 誘導時，K562細胞走向巨核球系分化。過去實驗室研究發現蛋白精胺酸甲基轉移酶1 (protein arginine methyltransferase 1, PRMT1) 能經由選擇性活化p38α來抑制巨核球細胞系分化，但是PRMT1如何調控p38 MAPK活化目前尚不清楚。文獻指出p38 MAPK可由上游分子MAPKK、MAPKKK及small G protein，如Rho家族調控。而RhoGDIα (Rho GDP-dissociation factor ?? 可抑制Rho GTPases家族的活化，並且文獻報導指出RhoGDIα含有三個會被甲基化的精胺酸。因此本研究探討RhoGDIα是否參與調控巨核球分化，以及其甲基化可能的影響。
本研究以劉氏染色法，發現RhoGDIα可促進K562的巨核球分化，並在RhoGDIα缺失穩定細胞株中發現磷酸化p38增加。SB203580 (p38抑制劑) 則回復RhoGDIα缺失所降低的分化。而當p38α缺失時，RhoGDIα對分化的影響則消失，顯示RhoGDIα經由p38α MAPK調控巨核球分化。並且研究發現PRMT1可部分回復由RhoGDIα促進巨核球分化的現象。細胞中RhoGDIα的第111個及第152個精胺酸曾被報導可被甲基化，突變第111個或第152個精胺酸會降低部分RhoGDIα促進的巨核球分化。本研究為首次發現RhoGDIα參與調控巨核球系分化的角色。

The mitogen-activated protein kinase (MAPK) pathways regulate various cellular functions, including differentiation. Activation of Erk MAPK pathway promotes megakaryocytic (MK) differentiation and p38 MAPK pathway plays a negative role in MK differentiation. K562 is a multipotent human leukemia cell line which can be induced to differentiate into various lineages including megakaryocytes. Upon PMA (phorbol 12-myristate 13-acetate) treatment, K562 cells display characteristics of MK and are often used as a model cell line. Our previous study demonstrated that PRMT1 (protein arginine methyltransferase 1) inhibited PMA-induced MK differentiation of K562 cells via activation of p38? MAPK. However, the molecular mechanism is unclear. The activity of the p38 MAPK pathway is regulated by the upstream MAPKK and MAPKKK and small G proteins such as Rho family. The Rho GDP-dissociation factor ? (RhoGDI?? is known to inhibit activity of Rho GTPases and has been shown to contain three arginine residues that can be dimethylated.
This study showed that RhoGDI? plays a positive role in PMA-induced MK differentiation of K562 cells as demonstrated by changes in cytological characteristics. Activation of p38 was enhanced in RhoGDI? knockdown cells by Western blot analysis. Suppression of MK differentiation in RhoGDI? knockdown cells was reverted upon treatment of p38 inhibitor (SB203580). RhoGDI? could reverse the MK differentiation in p38β knockdown cells but not in p38? knockdown cells. In context with PRMT1, promotion of MK differentiation by RhoGDI? was reversed. Mutations at Arg111 or Arg152, which are known to be methylated in cells, abolished the promotion of MK differentiation by RhoGDI?? Together, our results suggest, for the first time, that RhoGDI? and the potential role of methylation regulates PMA-induced megakaryocytic differentiation of K562.

目 錄 ………………………………………………………… 1
縮 寫 表 ………………………………………………………… 2
圖次目錄 ………………………………………………………… 4
附圖目錄 ………………………………………………………… 4
中文摘要 ………………………………………………………… 5
英文摘要 ………………………………………………………… 6
緒 論 ………………………………………………………… 7
研究目標 ………………………………………………………… 16
實驗材料 ………………………………………………………… 17
實驗方法 ………………………………………………………… 21
結 果 ………………………………………………………… 29
討 論 ………………………………………………………… 36
參考文獻 ………………………………………………………… 39
圖 表 ………………………………………………………… 47
附 圖 ………………………………………………………… 58

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