A study on the deleterious effect of dexamethasone on human tendon fibroblast and possible rescue effect of platelet-derived growth factor isoform B (PDGFBB).

Tang Yin Nei.Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.Includes bibliographical references (leaves xv-xxv).Abstracts in English and Chinese.ACKNOWLEDGEMENT --- p.iABBREVIATIONS --- p.ii-iiiINDEX FOR FIGURES --- p.iv-vINDEX FOR TABLES --- p.viABSTRACT (Chinese and English) --- p.vii-xiTABLE OF CONTENTS --- p.xii-xivChapter CHAPTER I 226}0ؤ --- INTRODUCTION --- p.1Chapter 1.1 --- Background --- p.2Chapter 1.2 --- TendonChapter 1.2.1 --- Structure and function --- p.3Chapter 1.2.2 --- Tendon fibroblast --- p.6Chapter 1.2.3 --- Components of the extracellular matrix --- p.7Chapter 1.2.3.1 --- Collagen --- p.8Chapter 1.2.3.2 --- Proteoglycan --- p.9Chapter 1.2.3.3 --- Non-collagenous structural glycoprotein --- p.10Chapter 1.3 --- Inflammation disorders of tendonChapter 1.3.1 --- Inflammation --- p.11Chapter 1.3.2 --- Treatment --- p.12Chapter 1.3.2.1 --- Glucocorticoid as an anti-inflammatory agent --- p.12Chapter 1.3.2.2 --- Dexamethasone --- p.14Chapter 1.3.3 --- Clinical occurrence of tendon rupture --- p.15Chapter 1.3.4 --- Animal research related to glucocorticoids and tendon rupture --- p.18Chapter 1.4 --- Platelet-derived growth factor isoform B (PDGFBB)Chapter 1.4.1 --- Structure and function --- p.21Chapter 1.4.2 --- PDGFbb effects on connective tissue --- p.22Chapter CHAPTER II 226}0ؤ --- AIM OF THE STUDY --- p.23Chapter 2.1 --- Limitations of the past researches --- p.24Chapter 2.2 --- Hypothesis of this study --- p.25Chapter 2.3 --- Objectives --- p.26Chapter 2.4 --- Long term significance --- p.26Chapter CHAPTER III 226}0ؤ --- METHODOLOGY --- p.27Chapter 3.1 --- Chemicals and materials usedChapter 3.1.1 --- Chemicals --- p.28Chapter 3.1.2 --- Materials --- p.28Chapter 3.2 --- Specimen collection and preparationChapter 3.2.1 --- Collection --- p.29Chapter 3.2.2 --- Preparation and isolation --- p.30Chapter 3.2.3 --- Cell culture --- p.31Chapter 3.3 --- Reagent preparationChapter 3.3.1 --- Charcoal-stripped serum --- p.32Chapter 3.3.2 --- Phenol-red free DMEM --- p.33Chapter 3.3.3 --- MTT --- p.33Chapter 3.3.4 --- Dexamethasone --- p.34Chapter 3.3.5 --- PDGFbb --- p.34Chapter 3.3.6 --- Trypan blue --- p.35Chapter 3.3.7 --- TCA/Tannic acid --- p.35Chapter 3.3.8 --- Collagenase buffer --- p.35Chapter 3.4 --- MorphologyChapter 3.4.1 --- Inverted phase contrast light microscopy --- p.36Chapter 3.4.2 --- Scanning electron microscopy --- p.36Chapter 3.5 --- Biological assaysChapter 3.5.1 --- "MTT (3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assay" --- p.38Chapter 3.5.1.1 --- Correlation between MTT assay and trypan blue dye method --- p.38Chapter 3.5.1.2 --- Growth kinetics for tendon fibroblasts --- p.41Chapter 3.5.1.3 --- Cell viability --- p.43Chapter 3.5.2 --- Brdu (5-bromo-2'-deoxyuridine) assay --- p.44Chapter 3.5.3 --- Flow cytometry --- p.45Chapter 3.5.4 --- Apoptosis --- p.47Chapter 3.5.5 --- 3H-Proline incorporation assay --- p.48Chapter 3.5.6 --- 35Sulfate incorporation assay --- p.51Chapter 3.5.7 --- Immunocytochemistry (PDGF-β receptor) --- p.54Chapter 3.6 --- Statistical analysisChapter 3.6.1 --- Dose-response curve of dexamethasone on cell viability and proliferation --- p.55Chapter 3.6.2 --- Comparison among various treatments of fibroblasts --- p.55Chapter CHAPTER I´Vؤ --- RESULTS --- p.56Chapter 4.1 --- In vitro effect of dexamethasone on rat tendon fibroblastsChapter 4.1.1 --- Viable cell number between two sexes --- p.57Chapter 4.2 --- In vitro effect of dexamethasone and PDGFBB on human tendon fibroblastsChapter 4.2.1 --- Gross morphology --- p.58Chapter 4.2.2 --- Cell cycle --- p.60Chapter 4.2.3 --- Apoptosis --- p.61Chapter 4.2.4 --- Viable cell numberChapter 4.2.4.1 --- Effect of dexamethasone --- p.62Chapter 4.2.4.2 --- Effect of PDGFBB --- p.63Chapter 4.2.5 --- Cell proliferationChapter 4.2.5.1 --- Effect of dexamethasone --- p.65Chapter 4.2.5.2 --- Effect of PDGFbb --- p.67Chapter 4.2.6 --- Collagen synthesis --- p.68Chapter 4.2.7 --- Proteoglycan synthesis --- p.72Chapter 4.2.8 --- PDGF-rβ expression --- p.74Chapter CHAPTER V 226}0ؤ --- DISCUSSION --- p.75Chapter 5.1 --- Dexamethasone and PDGFBB induced change of cell morphology --- p.77Chapter 5.2 --- Dexamethasone retarded cell growth of human tendon fibroblast --- p.80Chapter 5.3 --- Dexamethasone inhibited collagen synthesis --- p.82Chapter 5.4 --- Dexamethasone inhibited proteoglycan synthesis --- p.86Chapter 5.5 --- PDGFbb could counteract the inhibitory effects of dexamethasone --- p.88Chapter 5.6 --- Expression of PDGF-(3 receptor is regulated by dexamethasone and PDGFBB --- p.90Chapter 5.7 --- Limitations of this studyChapter 5.7.1 --- Not enough sample to differentiate different between two sexes --- p.92Chapter 5.7.2 --- Small sample size and few assays --- p.92Chapter 5.7.3 --- Limitations of the cell culture model --- p.93Chapter 5.7.4 --- Difficult to further in vivo study on human --- p.93Chapter 5.8 --- Contributions of this studyChapter 5.8.1 --- Improve the limitation of the past research --- p.94Chapter 5.8.1.1 --- Human tendon specimen --- p.94Chapter 5.8.1.2 --- In vitro system --- p.94Chapter 5.8.2 --- Understand the effect of dexmaethasone on human tendon fibroblasts --- p.95Chapter 5.8.3 --- Counteract the deleterious effects of dexamethasone by PDGFBB --- p.95Chapter CHAPTER VÍؤ --- CONCLUSION & FUTURE STUDY --- p.96Chapter 6.1 --- Conclusion --- p.97Chapter 6.2 --- Future study --- p.98Chapter 6.2.1 --- Study the balance between matrix synthesis and degradation --- p.98Chapter 6.2.2 --- Determine collagen typing --- p.99Chapter 6.2.3 --- Further explore the effect of glucocorticoid in organ culture model --- p.100Chapter 6.2.4 --- Investigate molecular mechanism of dexamethasone and PDGFBB --- p.100REFERENCES --- p.xv-xxvAPPENDIX --- p.xxv