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分析基因(KLF1、KLF13和E2F2)在紅血球分化 可能扮演的角色

Potential roles of genes (KLF1, KLF13, and E2F2) associated with erythrocytic differentiation

作者:陳仁毅
畢業學校:慈濟大學
出版單位:慈濟大學
核准日期:2012-10-04
類型:Electronic Thesis or Dissertation
權限:Copyright information available at source archive--Tzu Chi University....

中文摘要

紅血球的生成是一個透過許多調控機制,多變複雜且需要歷經多個階段性的過程,我們對於紅血球分化的過程與分子機轉至今尚未完全清楚。K562細胞是來自於人類慢性骨髓性白血病患者所建立的細胞株,透過不同的藥物處理,可以誘導分化為紅血球或巨核細胞。我們實驗室之前的研究顯示,炭疽桿菌 (Bacillus anthracis)致死毒素 (lethal toxin)能夠抑制給予GTP(guanosine 5’-triphosphate)處理之後所誘導的K562細胞株往紅血球分化。我們利用基因晶片方式,來瞭解那些特定的基因對於紅血球分化的調控上具有相關性。由初步結果中挑選出3個候選基因,分別為KLF1 (Krüppel-like factor 1; Erythroid Krüppel-like transcription factor, EKLF), KLF13 (Krüppel-like factor 13) 和 E2F2 (E2F transcription factor 2),這些基因在給予GTP處理誘導分化後表現量增加,並在前處理LT 2小時再給予GTP處理誘導分化後表現量減少。我們也利用定量逆轉錄聚合酶鏈鎖反應 (qRT-PCR),來證明這些基因在給予GTP處理誘導分化後表現量亦會增加。我們假設這些基因可能在正常紅血球分化和受LT抑制紅血球分化的過程中,可能扮演關鍵重要的角色。在本篇研究中,我們利用shRNA干擾方式knockdown K562細胞的KLF1、KLF13和E2F2基因之mRNA,再給予GTP誘導其分化,藉由流式細胞儀分析發現其紅血球表面抗原 (GPA)表現量降低,細胞往紅血球分化受到抑制。進一步,我們也建立以人類臍帶血而來的CD34+造血幹細胞,透過in vitro differentiation往紅血球分化的模式,了解在分化過程中,這3個候選基因表現的情況。最後,我們利用shRNA方式降解 CD34+細胞的KLF1和E2F2基因的mRNA表現量,也觀察到會更促進紅血球分化;而在降解 CD34+細胞的KLF13基因的mRNA表現量,則會抑制其往紅血球分化。在本篇研究中,我們證明KLF1、KLF13和E2F2基因在紅血球分化過程中可能扮演重要的角色。

英文摘要

Erythropoiesis is a multi-stage process regulated by many specific genes, but the underlying mechanisms are still unknown. K562 a cell line derived from human myelogenous leukaemia; it can go through erythrocytic or megakaryocytic lineages differentiation upon treatments with different inducers. Our previous studies demonstrated that Bacillus anthracis lethal toxin (LT) could suppress GTP (guanosine 5 -triphosphate) induced erythrocytic differentiation in K562 cells. To investigate the novel genes that may regulate erythrocytic differentiation, microarray analysis was performed. Our data indicated that three genes: KLF1 (Krüppel-like factor 1; Erythroid krueppel-like transcription factor, EKLF), KLF13 (Krüppel-like factor 13), and E2F2 (E2F transcription factor 2) were up-regulated after GTP treatments and down-regulated upon LT pre-treatments. We hypothesized that these genes may play major roles in normal and LT-suppressed erythrocytic differentiation. In this study, we have confirmed that all three genes were up-regulated upon GTP treatments by quantitative reverse transcription polymerase chain reaction (qRT-PCR). When each of three genes was knockdown by short hairpin-mediated RNAs (shRNAs) in K562 cells and suppressed the expression of erythrocytic specific surface marker (glycophorin A) monitored by flow cytometry. Further, we established an in vitro erythrocytic differentiation system by using CD34+ hematopoietic stem cells (HSCs), which are enriched from human unbilical cord blood. The dynamic expression and functional roles of these candidate genes during erythrocytic differentiation were verified in cord blood derived hematopoietic stem cells. In this study, we demonstrated that KLF1, E2F2, and KLF13 could affect erythrocytic differentiation through RNA interference experiments. In conclusion, these results suggest KLF1, KLF13, and E2F2 genes play potential roles in erythrocytic differentiation.


指導教授 - 孫德珊


 

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