Role of reactive oxygen species (ROS) in cardiomyocyte differentiation of mouse embryonic stem cells.

Law, Sau Kwan.Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.Includes bibliographical references (leaves 111-117).Abstract also in Chinese.Thesis Committee --- p.iAcknowledgements --- p.iiContents --- p.iiiAbstract --- p.vii論文摘要 --- p.xAbbreviations --- p.xiList of Figures --- p.xiiiList of Tables --- p.xxiiiChapter CHAPTER ONE --- INTRODUCTIONChapter 1.1 --- Embryonic Stem (ES) CellsChapter 1.1.1 --- Characteristics of ES Cells lChapter 1.1.2 --- Therapeutic Potential of ES Cells --- p.3Chapter 1.1.3 --- Myocardial Infarction and ES cells-derived Cardiomyocytes --- p.4Chapter 1.1.4 --- Current Hurdles of Using ES cells-derived Cardiomyocytes for Research and Therapeutic Purposes --- p.6Chapter 1.2 --- Transcription Factors for Cardiac DevelopmentChapter 1.2.1 --- GATA-binding Protein 4 (GATA-4) --- p.8Chapter 1.2.2 --- Myocyte Enhancer Factor 2C (MEF2C) --- p.10Chapter 1.2.3 --- "NK2 Transcription Factor Related, Locus 5 (Nkx2.5)" --- p.11Chapter 1.2.4 --- Heart and Neural Crest Derivatives Expressed 1 /2 (HANDI/2) --- p.11Chapter 1.2.5 --- T-box Protein 5 (Tbx5) --- p.13Chapter 1.2.6 --- Serum Response Factor (SRF) --- p.14Chapter 1.2.7 --- Specificity Protein 1 (Spl) --- p.15Chapter 1.2.8 --- Activator Protein 1 (AP-1) --- p.16Chapter 1.3 --- Reactive Oxygen Species (ROS)Chapter 1.3.1 --- Cellular Production of ROS --- p.18Chapter 1.3.2 --- Maintenance of Redox balance --- p.18Chapter 1.3.3 --- Redox Signaling --- p.19Chapter 1.4 --- Nitric Oxide (NO) and NO Signaling --- p.20Chapter 1.5 --- Aims of the Study --- p.22Chapter CHAPTER TWO --- MATERIALS AND METHODSChapter 2.1 --- Mouse Embryonic Fibroblast (MEF) CultureChapter 2.1.1 --- Derivation of MEF --- p.23Chapter 2.1.2 --- Maintenance of MEF Culture --- p.24Chapter 2.1.3 --- Irradiation of MEF --- p.25Chapter 2.2 --- Mouse ES Cell CultureChapter 2.2.1 --- Maintenance of Undifferentiated Mouse ES Cell Culture --- p.26Chapter 2.2.2 --- Differentiation of Mouse ES Cells --- p.26Chapter 2.2.3 --- Exogenous addition of hydrogen peroxide (H2O2) and NO --- p.27Chapter 2.3 --- ROS Localization StudyChapter 2.3.1 --- Frozen Sectioning --- p.28Chapter 2.3.2 --- Confocal microscopy for ROS detection --- p.28Chapter 2.4 --- Intracellular ROS MeasurementChapter 2.4.1 --- "Chemistry of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)" --- p.29Chapter 2.4.2 --- Flow Cytometry for ROS Measurement --- p.29Chapter 2.5 --- Gene Expression StudyChapter 2.5.1 --- Primer Design --- p.30Chapter 2.5.2 --- RNA Extraction --- p.31Chapter 2.5.3 --- DNase Treatment --- p.32Chapter 2.5.4 --- Reverse Transcription --- p.32Chapter 2.5.5 --- Quantitative Real Time PCR --- p.33Chapter 2.5.6 --- Quantification of mRNA Expression --- p.34Chapter 2.6 --- Protein Expression StudyChapter 2.6.1 --- Total Protein Extraction --- p.34Chapter 2.6.2 --- Nuclear and Cytosolic Protein Extraction --- p.35Chapter 2.6.3 --- Measurement of Protein Concentration --- p.36Chapter 2.6.4 --- De-sumoylation Assay --- p.36Chapter 2.6.5 --- De-phosphorylation Assay --- p.37Chapter 2.6.6 --- De-glycosylation Assay --- p.38Chapter 2.6.7 --- Western Blot --- p.39Chapter 2.7 --- Statistical Analysis --- p.41Chapter CHAPTER THREE --- RESULTSChapter 3.1 --- Study of Endogenous ROSChapter 3.1.1 --- Level and Distribution of Endogenous ROS --- p.47Chapter 3.1.2 --- Quantification of intracellular ROS --- p.48Chapter 3.2 --- Effect of Exogenous Addition of Nitric Oxide (NO) on Cardiac DifferentiationChapter 3.2.1 --- Beating Profile of NO-treated Embryoid Bodies (EBs) --- p.50Chapter 3.3 --- Effect of Exogenous Addition of H2O2 on Cardiac DifferentiationChapter 3.3.1 --- Beating Profile of H2O2-treated EBs --- p.51Chapter 3.3.2 --- mRNA Expression of Cardiac Structural Genes --- p.52Chapter 3.3.3 --- Protein Expression of Cardiac Structural Genes --- p.54Chapter 3.3.4 --- mRNA Expression of Cardiac Transcription Factors --- p.58Chapter 3.3.5 --- Protein Expression of Cardiac Transcription Factors --- p.67Chapter 3.3.6 --- Post-translational Modifications of Cardiac Transcription Factors --- p.74Chapter 3.3.7 --- Translocation of Cardiac Transcription Factors --- p.89Chapter CHAPTER FOUR --- DISCUSSIONChapter 4.1 --- Changes in the Level of Endogenous ROS During Cardiac Differentiation of Mouse ES Cells --- p.96Chapter 4.2 --- H2O2 and NO Have Opposite Effects Towards Cardiac Differentiation --- p.97Chapter 4.3 --- Exogenous Addition of H2O2 Advances Differentiation of Mouse ES Cells into Cardiac Lineage --- p.99Chapter 4.4 --- Possible Role of H2O2 in Mediating Cardiac Differentiation of Mouse ES Cells --- p.103Chapter 4.5 --- Future Directions --- p.108Conclusions --- p.110References --- p.11