Leung, Sze Lee Cecilia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 144-153). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / Abbreviations --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.1.1 --- Adult stem cells --- p.2 / Chapter 1.1.2 --- Embryonic stem cells --- p.2 / Chapter 1.1.3 --- Pros and cons of embryonic and adult stem cells --- p.5 / Chapter 1.1.4 --- Human embryonic stem cells (hESCs) --- p.6 / Chapter 1.1.5 --- Mouse embryonic stem cells (mESCs) --- p.7 / Chapter 1.1.6 --- Characteristics of ESC-derived cardiomyocytes --- p.7 / Chapter 1.2 --- Cardiovascular Diseases (CVD) --- p.9 / Chapter 1.2.1 --- Causes and statistics of CVD --- p.9 / Chapter 1.2.2 --- Current treatment for CVD --- p.10 / Chapter 1.2.3 --- Current hurdles of putting hESC-CMs into clinical use --- p.11 / Chapter 1.3 --- Myosin light chain2v --- p.13 / Chapter 1.4 --- Genetic-engineering of hESCs & their cardiac derivatives by lentiviral-mediate gene transfer --- p.14 / Chapter 1.5 --- Cytokines secretion during myocardial infarction --- p.15 / Chapter 1.6 --- Aims of the Project --- p.19 / Chapter 1.7 --- Significance of the Project --- p.19 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Subcloning --- p.20 / Chapter 2.1.1 --- Amplification of MLC-2v --- p.20 / Chapter 2.1.2 --- Purification of DNA product --- p.21 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.21 / Chapter 2.1.4 --- Ligation of MLC-2v promoter with DuetO 11 vector --- p.22 / Chapter 2.1.5 --- Transformation of ligation product into competent cells --- p.22 / Chapter 2.1.6 --- PCR confirmation of successful ligation --- p.23 / Chapter 2.1.7 --- Small-scale preparation of bacterial plasmid DNA --- p.23 / Chapter 2.1.8 --- Restriction enzyme digestions to reconfirm positive clones --- p.24 / Chapter 2.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.25 / Chapter 2.1.10 --- Large-scale preparation of target recombinant expression vector --- p.25 / Chapter 2.2 --- Mouse Embryonic Fibroblast (MEF) Culture --- p.26 / Chapter 2.2.1 --- Derivation of MEF --- p.26 / Chapter 2.2.2 --- Mouse embryonic fibroblast cells culture --- p.27 / Chapter 2.2.3 --- Irradiation of mouse embryonic fibroblast --- p.28 / Chapter 2.3 --- HESC culture --- p.29 / Chapter 2.3.1 --- Thawing and Plating hESCs --- p.29 / Chapter 2.3.2 --- Splitting hESCs --- p.30 / Chapter 2.3.3 --- "Culture maintainence, selection and colony removal" --- p.31 / Chapter a) --- Distinguish differentiated and undifferentiated cells and colonies / Chapter b) --- "Remove differentiated cells by ""Picking to Remove""" / Chapter c) --- "Remove undifferentiated cells by ""Picking to Keep""" / Chapter 2.3.4 --- Freezing hESCs --- p.31 / Chapter 2.3.5 --- Differentiation of hESCs --- p.32 / Chapter 2.3.6 --- "HESC culture on feeder free system, mTeSR TM1" --- p.34 / Chapter a) --- Preparation of mTeSRTMl / Chapter b) --- Preparation of BD MatrigelTM hESC-qualified Matrix aliquots / Chapter c) --- Coating plates with BD MatrigelTM hESC-qualified Matrix / Chapter d) --- Human Embryonic stem cells culture in mTeSRTMl / Chapter 2.4 --- ES Cell Characterization (Chemicon Cat# SCR001) --- p.36 / Chapter 2.4.1 --- Alkaline Phosphatase Staining --- p.36 / Chapter 2.4.2 --- Immunofluorescence staining --- p.37 / Chapter 2.5 --- MESC culture --- p.38 / Chapter 2.5.1 --- Thawing and Plating mESCs --- p.38 / Chapter 2.5.2 --- Splitting mESCs --- p.38 / Chapter 2.5.3 --- Differentiation of mESCs --- p.39 / Chapter 2.5.4 --- To study the effects of cytokines on mESC differentiation --- p.40 / Chapter 2.6 --- Lentivirus (LV) Packaging --- p.41 / Chapter 2.6.1 --- Transfection of lentiviral vectors into HEK293FT cells --- p.41 / Chapter 2.6.2 --- LV titering --- p.42 / Chapter 2.7 --- MultipleTransduction --- p.43 / Chapter 2.8 --- Selection of transduced cells by hygromycin --- p.43 / Chapter 2.8.1 --- Determination of hygromycin selection dosage --- p.43 / Chapter 2.8.2 --- Selection of stable clones --- p.44 / Chapter 2.9 --- Isolation of green fluorescent cardiomyocytes derived from differentiated hESCs --- p.45 / Chapter 2.9.1 --- Collagenase digestion of embryoid bodies into single cells --- p.45 / Chapter 2.9.2 --- FACS --- p.46 / Chapter 2.10 --- Gene expression study / Chapter 2.10.1 --- Primer design --- p.46 / Chapter 2.10.2 --- RNA extraction --- p.46 / Chapter 2.10.3 --- DNase Treatment --- p.47 / Chapter 2.10.4 --- Synthesis of Double-stranded cDNA from Total RNA --- p.47 / Chapter 2.10.5 --- Quantitative real-time PCR --- p.48 / Chapter 2.10.6 --- Quantification of mRNA expression --- p.49 / Chapter 2.11 --- Protein Expression study --- p.49 / Chapter 2.11.1 --- Crude protein extraction --- p.49 / Chapter 2.11.2 --- Quantitation of protein samples --- p.50 / Chapter 2.11.3 --- SDS-PAGE --- p.50 / Chapter 2.11.4 --- Western Blot --- p.51 / Chapter 2.11.5 --- Western blot luminal detection --- p.52 / Chapter 2.11.6 --- Quantification of protein expression --- p.52 / Chapter CHAPTER 3 --- PURIFICATION OF CARDIOMYOCYTES DERIVED FROM DIFFERENTIATED HESCs / Chapter 3.1 --- Subcloning --- p.57 / Chapter 3.1.1 --- Linearization of DuetO11 and excision of UBC promoter --- p.58 / Chapter 3.1.2 --- PCR cloning of MLC-2V --- p.59 / Chapter 3.1.3 --- Ligation of MLC-2v promoter to linearized DuetO11 --- p.60 / Chapter 3.1.3.1 --- Colony PCR to screen for positive clones --- p.61 / Chapter 3.1.3.2 --- Restriction digestion to confirm the success of ligation --- p.61 / Chapter 3.2 --- Lentivirus (LV) packaging --- p.62 / Chapter 3.2.1 --- Transfection --- p.63 / Chapter 3.2.2 --- LV titering --- p.64 / Chapter 3.3 --- HESC culture --- p.66 / Chapter 3.4 --- Multi-transduction of hESCs with LVs --- p.67 / Chapter 3.5 --- Differentiation after transduction --- p.69 / Chapter 3.6 --- Antibiotic selection --- p.71 / Chapter 3.6.1 --- Characterization of hESCs on feeder free system --- p.72 / Chapter 3.6.1.1 --- Alkaline Phosphatase (AP) staining --- p.72 / Chapter 3.6.1.2 --- Immunostaining with pluripotency marker --- p.73 / Chapter 3.6.2 --- Determination of hygromycin dosage by MTT assay --- p.74 / Chapter 3.6.3 --- HESCs after selection in feeder free system --- p.75 / Chapter 3.7 --- Differentiation of hESCs after selection --- p.76 / Chapter 3.8 --- FACS --- p.77 / Chapter 3.9 --- QPCR of cells after FACS --- p.80 / Chapter 3.9.1 --- Gene expression of Nkx2.5 --- p.81 / Chapter 3.9.2 --- Gene expression of c-Tnl --- p.82 / Chapter 3.9.3 --- Gene expression of c-TnT --- p.83 / Chapter 3.9.3 --- Gene expression of MLC-2v --- p.84 / Chapter CHAPTER 4 --- THE STUDY OF CYTOKINES' EFFECT ON MESC DIFFERENTIATION / Chapter 4.1 --- mESC culture --- p.85 / Chapter 4.2 --- The effect of cytokines on the differentiation of mESCs --- p.86 / Chapter 4.2.1 --- Beating curves of mESCs treated with different concentrations of cytokines at differentiation day 2 to 6 before attachment --- p.88 / Chapter 4.2.2 --- qPCR to determine the cytokines' effect on the differentiation of mESCs --- p.94 / Chapter 4.2.2.1 --- The effect of IL-1α on the expression of cardiac specific genes --- p.95 / Chapter 4.2.2.2 --- The effect of IL-1β on the expression of cardiac specific genes --- p.98 / Chapter 4.2.2.3 --- The effect of IL-6 on the expression of cardiac specific genes --- p.101 / Chapter 4.2.2.4 --- The effect of IL-10 on the expression of cardiac specific genes --- p.104 / Chapter 4.2.2.5 --- The effect of IL-18 on the expression of cardiac specific genes --- p.107 / Chapter 4.2.2.6 --- The effect of TNF-α on the expression of cardiac specific genes --- p.110 / Chapter 4.2.3 --- Western blot analysis of the cytokines' effect on the differentiation of mESCs --- p.113 / Chapter 4.2.3.1 --- The effect of IL-lα on the abundance of cardiac specific proteins --- p.114 / Chapter 4.2.3.2 --- The effect of IL-1β on the abundance of cardiac specific proteins --- p.116 / Chapter 4.2.3.3 --- The effect of IL-6 on the abundance of cardiac specific proteins --- p.118 / Chapter 4.2.3.4 --- The effect of IL-10 on the abundance of cardiac specific proteins --- p.120 / Chapter 4.2.3.5 --- The effect of IL-18 on the abundance of cardiac specific proteins --- p.122 / Chapter 4.2.3.6 --- The effect of TNF-α on the abundance of cardiac specific proteins --- p.124 / Chapter CHAPTER 5 --- DISCUSSION / Chapter 5.1 --- Purification of cardiomyocytes derived from differentiated hESCs --- p.127 / Chapter 5.2 --- Study on the effect of cytokines on mESC differentiation --- p.135 / Chapter 5.3 --- Conclusion --- p.142 / REFERENCES --- p.144
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326543 |
| Date | January 2008 |
| Contributors | Leung, Sze Lee Cecilia., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvi, 153 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
Page generated in 0.0032 seconds