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研究生:Agustina Bungahot
研究生(外文):AGUSTINA BUNGAHOT
論文名稱(外文):Exploration of tyrosine kinase nhibitor-mediated resistance in chronic myeloid leukemia
指導教授:尤仁音
指導教授(外文):YOU, REN-IN
口試委員(外文):SUN, DER-SHANHSU, JUNG-HSIN
口試日期:2017-07-17
學位類別:碩士
校院名稱:慈濟大學
系所名稱:醫學生物技術碩士班
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:2017
畢業學年度:105
語文別:英文
論文頁數:45
外文關鍵詞:chronic myeloid leukemia
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Chronic Myelogenous Leukemia (CML) is a hematologic malignancy that diagnosed by BCR-ABL transcript levels, cytogenetic response and bone marrow blast cells. Tyrosine kinase inhibitors (TKIs) act specifically and effectively at BCR-ABL to promote the treatment of CML cancer cells as the first line target therapy. However, some TKIs are ineffective against Ph+ ALL or CML. Aberrant epigenetic markers have been considered as a critically contributor to the acquisition of chemotherapeutics and targeted cancer drugs resistance. To investigate the underlying mechanism of TKI targeted cancer drugs resistance, we conducted knockdown epigenetic related genes (Fem1b, RPRM, and MLH1) in K562 parental cells. We analyzed cell growth, signaling pathways, cell proliferation and differentiation assay to validate the alternative pathways to confer imatinib resistance. The cell cycle of K562 parental and derived cells were no different. But the level of pErk was sustained only in MLH1 KD cells. We also found that the expression level of PERK and eIF2α were decreased in the knockdown cells. This result shows that ER stress might involve in imatinib-mediated resistance in CML. We further established K562 cells expressed with epigenetic modifier, and evaluate the expression of those epigenetic targets. The expression level of one of epigenetic related gene, Evi1 gene, was increased in imatinib-resistant cells. To investigate the role of this gene in imatinib-resistant cells, we overexpressed the expression of Evi1 in K562 parental cells. We analyzed the sensitivity of the Evi1 OE cells to imatinib and we found that the sensitivity of the cells will decreased when the expression of Evi1 was increased.
We further investigated the differentiation potency of these stable clones using hemin and PMA stimulation in K562 parental cells and epigenetic related genes-knockdown cells. In addition to elucidate TKI drug response of these cell clones, the tumor xenograft was performed. The setup platforms are suitable to exam the important regulators to drive TKI resistance.
Abstract
Table of contents
Chapter I. Introduction 1
Chapter II. Aim of study 8
Chapter III. Material and methods 10
1. Cell lines 10
2. Cell Transfection 10
3. MTT assay 10
4. Cell cycle analysis 11
5. Western blot analysis 11
6. Antibodies 11
7. Xenograft for tumor growth model 12
8. Cell differentiation 12
9. Surface marker staining 12
Chapter IV Results 13
1. Tyrosine kinase inhibitor-resistant related genes in CML 13
2. The cytotoxicity effect of imatinib on K562 parental and derived cells 14
3. Effect of imatinib on the signaling pathways 15
4. Regulation of the Fem1b Expression 16
5. The effect of imatinib on Evi1-OE cells 17
6. Differentiation cells 18
6.1 Morphology: The differentiation of K562 to erythrocyte 18
6.2 Hemoglobin concentration: The differentiation of K562 to
erythrocyte 18
6.3 Surface marker: The differentiation of K562 to erythrocyte 18
6.4 Morphology: The differentiation of K562 to megakaryocyte 19
7. Tumor growth of K562 cells in vivo 19
Chapter V. Discussion 20
References 23
Figures 31
1. Establishment of K562 cells knockdown of Fem1b, RPRM, and MLH1 31
2. Effect of Imatinib on K562 and derived cell clones 33
3. Effect of Imatinib on the level of phosphorylated ERK 34
4. Expression of PERK and eIF2α in K562 cells and stable clones 35
5. Characterization of Evi1 overexpressed cells 36
6. Effect of Imatinib on cell cycle in Evi1 overexpressed cells 37
7. Proliferation rate of K562 cells 38
8. The differentiation of K562 cells to erythrocyte 39
9. Hemoglobin level in hemin-induced K562 cell 41
10. Expression level of CD235 in hemin-induced K562 cells differentiation to erythrocyte 42
11. The differentiation of K562 cells to megakaryocyte 43
12. K562 xenograft to NOD-SCID mice 45
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