The role of reactive oxygen species during erythropoiesis: an in vitro model using TF-1 cells.

• Other Authors: Ge, Tianfang.
• Format: Others
• Language: English; Chinese
• Published: 2009
• Subjects: Erythropoiesis; Active oxygen; Reactive Oxygen Species
• Online Access: http://library.cuhk.edu.hk/record=b5894103; http://repository.lib.cuhk.edu.hk/en/item/cuhk-326891

Ge, Tianfang. === Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. === Includes bibliographical references (leaves 87-93). === Abstract also in Chinese. === EXAMINATION COMMITTEE LIST --- p.ii === DECLARATION --- p.iii === ACKNOWLEDGEMENTS --- p.iv === ABSTRACT --- p.v === ABSTRACT IN CHINESE --- p.vii === ABBREVIATIONS --- p.ix === TABLE OF CONTENTS --- p.xiii === Chapter 1 --- INTRODUCTION --- p.1 === Chapter 1.1 --- Erythropoiesis --- p.2 === Chapter 1.2 --- The TF-1 model --- p.3 === Chapter 1.3 --- The erythroid marker glycophorin A (GPA) --- p.4 === Chapter 1.4 --- Reactive oxygen species (ROS) --- p.4 === Chapter 1.5 --- Oxidative stress in human erythrocytes --- p.6 === Chapter 1.6 --- Antioxidant defense systems --- p.6 === Chapter 1.7 --- Glucose provides the majority of reducing equivalents in human erythrocytes --- p.9 === Chapter 1.8 --- Glucose transporter type 1 (Glut l) transports glucose and vitamin C into human erythrocytes --- p.10 === Chapter 1.9 --- Hypothesis and objectives --- p.11 === Chapter 1.10 --- Long-term significance --- p.12 === Figure 1.1 Stages of mammalian erythropoiesis. Adapted from (Koury et al.，2002) --- p.13 === "Figure 1.2 Conversion of major ROS. Adapted from (Ghaffari," --- p.14 === Figure 1.3 Major oxidative defense in human erythrocytes --- p.15 === "Figure 1.4 Peroxide scavenging systems. Adapted from (Day," --- p.16 === Chapter 2 --- MATERIALS AND METHODS --- p.17 === Chapter 2.1 --- Cell culture --- p.18 === Chapter 2.1.1 --- Culture media --- p.18 === Chapter 2.1.2 --- Cell maintenance --- p.19 === Chapter 2.1.3 --- Cell cryopreservation --- p.19 === Chapter 2.1.4 --- Cell differentiation --- p.20 === Chapter 2.1.5 --- Cell treatments --- p.20 === Chapter 2.1.5.1 --- Antioxidant treatments --- p.21 === Chapter 2.1.5.2 --- H2O2 challenging --- p.22 === Chapter 2.1.5.3 --- Antibiotic treatment --- p.22 === Chapter 2.2 --- Flow cytometry --- p.23 === Chapter 2.2.1 --- Flow cytometers --- p.23 === Chapter 2.2.2 --- Analysis of erythroid differentiation --- p.23 === Chapter 2.2.3 --- Analysis of cell lineage --- p.24 === Chapter 2.2.4 --- Analysis of intracellular ROS --- p.24 === Chapter 2.2.5 --- Analysis of mitochondrial transmembrane potential (Δψm) --- p.25 === Chapter 2.2.6 --- Analysis of mitochondrial mass --- p.25 === Chapter 2.2.7 --- Analysis of cell death --- p.26 === Chapter 2.2.8 --- Analysis of caspase-3 activity --- p.27 === Chapter 2.2.9 --- FACS cell sorting --- p.27 === Chapter 2.2.10 --- Two-variant flow cytometric experiments --- p.28 === Chapter 2.2.11 --- Analysis of flow cytometry data --- p.28 === Chapter 2.2.12 --- Compensation --- p.29 === Chapter 2.2.12.1 --- Compensation matrix for Annexin V-PI double-staining --- p.29 === Chapter 2.2.12.2 --- Compensation matrix for Annexin V-TMRM double-staining --- p.30 === Chapter 2.2.12.3 --- Compensation matrix for CFSE- GPA double-staining --- p.31 === Chapter 2.2.12.4 --- Compensation matrix for CFSE- TMRM double-staining --- p.31 === Chapter 2.2.12.5 --- Compensation matrix for CM- H2DCFDA-GPA double-staining --- p.32 === Chapter 2.2.12.6 --- Compensation matrix for GPA- TMRM double-staining --- p.33 === Chapter 2.3 --- Western blot --- p.35 === Chapter 2.4 --- Statistical analysis --- p.37 === Chapter 3 --- RESULTS AND DISCUSSION --- p.38 === Chapter 3.1 --- The cells with high GPA staining were younger in cell lineage --- p.39 === Chapter 3.2 --- ROS was produced during TF-1 erythropoiesis --- p.40 === Chapter 3.3 --- ROS production was not essential for TF-1 erythropoiesis --- p.41 === Chapter 3.4 --- ROS production was not the cause of cell proliferation during TF-1 erythropoiesis --- p.41 === Chapter 3.5 --- ROS production was not the cause of sub-lethal mitochondrial depolarization in TF-1 erythropoiesis --- p.42 === Chapter 3.6 --- The cells showing mitochondrial depolarization were mother cells that gave rise to differentiating cells --- p.44 === Chapter 3.7 --- ROS production was not the cause of cell death in TF-1 erythropoiesis --- p.45 === Chapter 3.8 --- ROS production confers oxidative defense during TF-1 erythropoiesis --- p.47 === Chapter 3.8.1 --- Glut l inhibition partially blocked TF-1 erythropoiesis without affecting cell viability --- p.47 === Chapter 3.8.2 --- Antioxidant defense systems were established during TF-1 erythropoiesis --- p.48 === Chapter 3.8.3 --- Antioxidant treatments blocked the establishment of antioxidant defense systems during TF-1 erythropoiesis --- p.51 === Chapter 3.9 --- Conclusion --- p.55 === Chapter 3.10 --- Future work --- p.56 === Figure 3.1 Cell lineage versus erythroid marker during erythropoiesis under vitamin E treatment --- p.59 === Figure 3.2 ROS production during erythropoiesis --- p.60 === Figure 3.3 ROS production versus erythroid marker during erythropoiesis under vitamin E treatment --- p.61 === Figure 3.4 Percentage of ROS+ cells in vitamin E-treated TF-1 erythropoiesis as compared to control --- p.63 === Figure 3.5 Percentage of GPA+ cells in vitamin E-treated TF-1 erythropoiesis as compared to control --- p.64 === Figure 3.6 Cell death versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under vitamin E treatment --- p.65 === Figure 3.7 Erythroid marker versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under vitamin E treatment --- p.67 === Figure 3.8 Cell lineage versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under vitamin E treatment --- p.69 === Figure 3.9 Change of mitochondrial mass during erythropoiesis --- p.71 === Figure 3.10 ROS production versus erythroid marker during erythropoiesis under levofloxacin treatment --- p.72 === Figure 3.11 Percentage of GPA+ cells in levofloxacin-treated TF-1 erythropoiesis as compared to control --- p.73 === Figure 3.12 Cell death versus mitochondrial transmembrane potential (Δψm) during erythropoiesis under levofloxac in treatment --- p.74 === Figure 3.13 Expression level of antioxidant enzymes during erythropoiesis --- p.75 === Figure 3.14 Expression level of Glut l during erythropoiesis --- p.76 === Figure 3.15 Expression level of Glut l in GPA positive and GPA negative populations --- p.77 === Figure 3.16 Cell death under oxidative stress challenging during erythropoiesis --- p.78 === Figure 3.17 Expression level of antioxidant enzymes and Glutl during erythropoiesis under EUK-134 treatment --- p.79 === Figure 3.18 Expression level of antioxidant enzymes and Glutl during erythropoiesis under vitamin E treatment --- p.80 === Figure 3.19 Cell death under oxidative stress challenging during erythropoiesis under vitamin E treatment --- p.82 === Figure 3.20 Expression level of antioxidant enzymes during erythropoiesis under vitamin C treatment --- p.83 === Figure 3.21 Cell death under oxidative stress challenging during erythropoiesis under vitamin C treatment --- p.84 === Figure 3.22 Cell death under oxidative stress challenging during erythropoiesis under NAC treatment --- p.85 === Figure 3.23 Summary of oxidative stress challenging during erythropoiesis --- p.86 === REFERENCES --- p.87