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研究生:廖俊哲
研究生(外文):Jun-Zhe Liao
論文名稱:探討Dsiabled-2影響sodiumbutyrate誘導K562細胞往紅血球分化的機制
論文名稱(外文):Functional analysis of Disabled-2 in sodium butyrate-induced erythroid differentiation of K562 cells
指導教授:曾慶平
指導教授(外文):Ching-Ping Tseng
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:64
中文關鍵詞:紅血球DAB2Sodium butyrate
外文關鍵詞:Erythroid differentiationDAB2Sodium butyrate
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Disabled-2 (DAB2)已知是腫瘤抑制基因(tumor suppressor gene)並且為一種接附蛋白(adaptor protein)可藉由和其他細胞內的蛋白質作用而傳遞下游訊息。本實驗室先前研究發現,當人類慢性骨髓性白血病細胞,K562被NaB誘導並分化成紅血球前驅細胞時,DAB2的表現量會上升,表示DAB2可能會調控這個分化過程。由於DAB2和紅血球分化之間的關係尚未釐清,因此本研究將探討DAB2在K562細胞藉由NaB誘導後往紅血球分化時所扮演的腳色。在以Benzidine staining比較K562,D-1-4,V7和可以穩定表現siDab2 siRNA的K562,si8的紅血球分化時發現,在DAB2表現被抑制的D-1-4及si8其紅血球分化的能力明顯受抑制(p<0.001),而此現象可以藉由重新表現human DAB2或p82後回復其往紅血球分化的能力(p<0.001)。此外也發現p59或B時可以被回復(p<0.001),然而表現p82 S24A 或 N時則無法回復其紅血球分化的能力。在Time course實驗中發現DAB2可藉由ERK-1/2調控紅血球分化,在重新表現human DAB2或p82時會抑制ERK-1/2而B無法調控ERK-1/2。雖然本研究發現Ser24 在DAB2調控紅血球分化時很重要,但其機制仍是未知。但本研究發現到ERK-1/2在DAB2調控紅血球分化時扮演次要的角色。
Disabled-2 (DAB2) has been known as a tumor suppressor gene and an adaptor protein that mediates various cellular signaling through interacting with other cellular proteins. Previous study in our lab found that DAB2 expression increased when human chronic myeloid leukemia cell line K562 was induced to differentiate into erythroid precursors by sodium butyrate (NaB), suggesting that DAB2 may regulate NaB-induced erythroid differentiation in K562. Since the relation of DAB2 to erythroid differentiation is still unclear, we plan to determine the role of DAB2 in NaB-induced erythroid differentiation. The erythroid differentiation of K562, D-1-4, V7, and si8 was determined by benzidine staining and found that erythroid differentiation of DAB2-lacking subclones, D-1-4 and si8, was inhibited (p<0.001); this can be rescued by overexpressing DAB2 by transfection of human DAB2 or p82 in D-1-4 and si8 (p<0.001). Furthermore, we found that p59 or B (p<0.001) can rescue erythroid differentiation of D-1-4 and si8; while p82 S24A or N can not rescue erythroid differentiation. Time-course experiment showed that ERK-1/2 may mediate DAB2-regulated erythroid differentiation. Overexpressing DAB2 and p82 inhibited ERK-1/2 phosphorylation while B can not modulate ERK-1/2 phosphorylation as other truncated DAB2. Although Ser24 is important for DAB2-regulated erythroid differentiation, the mechanism is still unknown in this study. However, ERK-1/2 phosphorylation was found to play part of role in NaB-induced erythroid differentiation in this study.
CHAPTER I. Introduction..........................................................................................................................1
A. Multi-functions of DAB2........................................................................................................................1
i. Discovery of DAB2.................................................................................................................................1
ii. Structure and function of DAB2.............................................................................................................1
iii. DAB2 signaling in cell differentiation....................................................................................................4
B. Sodium butyrate induces erythroid differentiation in K562 cells...........................................................6
i. Eryrthroid differentiation.........................................................................................................................6
ii. K562 cell................................................................................................................................................7
iii. Sodium butyrate....................................................................................................................................7
iv. Detection of erythroid differentiation......................................................................................................9
C. Study Aim............................................................................................................................................10
CHAPTER II. Materials and methods......................................................................................................11
A. Materials..............................................................................................................................................11
B. Plasmid construction...........................................................................................................................12
C. Cell culture and plasmid transfection..................................................................................................12
D. Purification of EGFP(+) cells by cell sorting........................................................................................13
E. Benzidine staining for detection of erythroid differentiation by sodium butyrate.................................13
F. Cell extract preparation and western blot............................................................................................14
G. Statistic analysis..................................................................................................................................14
CHAPTER III. Results..............................................................................................................................15
A. DAB2 expression affects NaB-induced erythroid differentiation..........................................................15
B. Construction of pUHD10-3 p82............................................................................................................15
C. DAB2 overexpression rescues erythroid differentiation of D-1-4 and si8............................................16
D. Construction of pUHD10-3 p59, p82 S24A, DB, and DN.....................................................................18
E. Ser24 phosphorylation site plays an important role in eryhtroid differentiation of D-1-4 and si8...........19
F. ERK1/2 phosphorylation may mediate DAB2-regulated erythroid differentiation..................................20
G. Modulation of DAB2 expression affects ERK1/2 phosphorylation in D-1-4 and si8 cells......................21
CHAPTER IV Discussion..........................................................................................................................22
REFERENCES.........................................................................................................................................27
Appendix...................................................................................................................................................36
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