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Dysregulation of retinoic acid synthesis in mouse embryos under diabetic or hyperglycemic conditions.

Chan, Wing Lung. / Thesis (M.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 111-130). / Abstracts in English and Chinese. / Title --- p.i / Acknowledgements --- p.ii / Table of Content --- p.iii / List of Tables --- p.viii / List of Figures --- p.xi / List of Graphs --- p.xii / Abbreviations --- p.xiv / Abstract --- p.xv / Abstract (Chinese) --- p.xvii / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Diabetes Mellitus --- p.2 / Chapter 1.1.1 --- Type 1 diabetes mellitus --- p.3 / Chapter 1.1.2 --- Type 2 diabetes mellitus --- p.4 / Chapter 1.1.3 --- Gestational diabetes mellitus --- p.5 / Chapter 1.2 --- Diabetic Pregnancy --- p.6 / Chapter 1.2.1 --- Incidence of congenital malformations in diabetic pregnancy --- p.6 / Chapter 1.2.2 --- Long term complications in the infant of diabetic mother --- p.7 / Chapter 1.3 --- Hyperglycemia --- p.7 / Chapter 1.4 --- Oxidative Stress --- p.8 / Chapter 1.4.1 --- Oxidative stress and antioxidant enzymes --- p.8 / Chapter 1.4.2 --- Cellular function of oxidative stress --- p.9 / Chapter 1.4.3 --- Adverse effects of excess oxidative stress during embryogenesis --- p.9 / Chapter 1.5 --- Retinoic Acid --- p.10 / Chapter 1.5.1 --- Function of RA during embryonic development --- p.10 / Chapter 1.5.2 --- RA synthesis and degradation --- p.10 / Chapter 1.5.3 --- Mechanisms of retinoic acid signaling : --- p.12 / Chapter 1.5.4 --- Developmental genes regulated by RA --- p.12 / Chapter 1.6 --- Strategy of the Thesis --- p.14 / Chapter Chapter 2: --- General Materials and Methods / Chapter 2.1 --- Animals --- p.17 / Chapter 2.2 --- Induction of Diabetes --- p.17 / Chapter 2.3 --- Mating Methods --- p.18 / Chapter 2.3.1 --- Mice --- p.18 / Chapter 2.3.2 --- Rats --- p.18 / Chapter 2.4 --- Whole Mount In Situ Hybridization --- p.19 / Chapter 2.4.1 --- Synthesis of DNA plasmids and riboprobes --- p.19 / Chapter 2.4.1.1 --- Mini-scale preparation of plasmid DNA --- p.19 / Chapter 2.4.1.2 --- Linearization of DNA plasmid --- p.20 / Chapter 2.4.1.3 --- In vitro transcription and labeling --- p.21 / Chapter 2.4.2 --- Fixation and dehydration of embryos --- p.22 / Chapter 2.4.3 --- Hybridization with RNA probes --- p.23 / Chapter 2.4.4 --- Post-hybridization wash --- p.24 / Chapter 2.4.4.1 --- Pre-absorption of anti-DIG antibody --- p.25 / Chapter 2.4.4.2 --- Embryo powder preparation --- p.25 / Chapter 2.4.5 --- Post antibody wash and signal development --- p.25 / Chapter 2.5 --- Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.26 / Chapter 2.5.1 --- Sample collection and storage --- p.26 / Chapter 2.5.2 --- Total RNA extraction --- p.27 / Chapter 2.5.3 --- Reverse transcription --- p.28 / Chapter 2.5.4 --- Quantitative real-time PCR --- p.28 / Chapter 2.5.5 --- Preparation of cDNA standards for real-time PCR --- p.29 / Chapter 2.6 --- RA-responsive Cell Line --- p.29 / Chapter 2.6.1 --- Cell culture --- p.30 / Chapter 2.6.2 --- Seeding 96-well plate with RA-responsive cells --- p.31 / Chapter 2.6.3 --- Applying samples to 96-well plate coated with RA-responsive cells --- p.31 / Chapter 2.6.4 --- β-galactosidase staining --- p.32 / Chapter 2.7 --- Separation of Protein Isoforms by Isoelectric Focusing (IEF) --- p.33 / Chapter 2.7.1 --- Preparing protein samples for IEF --- p.33 / Chapter 2.7.2 --- Isoelectric focusing --- p.33 / Chapter 2.7.3 --- IEF native gel staining --- p.34 / Chapter 2.7.4 --- Locating three retinaldehyde dehydrogenase (Raldh) isoforms --- p.35 / Chapter 2.8 --- In Vitro RA Synthesizing Reaction --- p.36 / Chapter Chapter 3: --- Effect of Maternal Diabetes on Retinoic Acid Synthesis in the Mouse Embryo / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Experimental Design --- p.41 / Chapter 3.3 --- Materials and Methods --- p.42 / Chapter 3.3.1 --- Sample collection --- p.42 / Chapter 3.3.1.1 --- Criteria for selecting embryos at the same developmental stage --- p.42 / Chapter 3.3.1.2 --- Sample collection for in situ hybridization --- p.42 / Chapter 3.3.1.3 --- Sample collection for real-time quantitative RT-PCR --- p.43 / Chapter 3.3.1.4 --- Sample collection for in vitro RA synthesizing reaction --- p.44 / Chapter 3.3.2 --- Statistical analyses --- p.45 / Chapter 3.4 --- Results --- p.46 / Chapter 3.4.1 --- "Comparison of the in situ expression pattern of Raldh 1, Raldh2 and Raldh3 between embryos of diabetic and non-diabetic mice" --- p.46 / Chapter 3.4.1.1 --- In situ hybridization patterns of Raldh 1 --- p.46 / Chapter 3.4.1.2 --- In situ hybridization patterns of Raldhl --- p.46 / Chapter 3.4.1.3 --- In situ hybridization patterns of Raldh3 --- p.47 / Chapter 3.4.2 --- "Comparison of the relative expression level of Raldh 1, Raldh2 and Raldh3 between embryos of diabetic and non-diabetic mice at different developmental stages" --- p.48 / Chapter 3.4.2.1 --- Relative expression levels of Raldh 1 --- p.50 / Chapter 3.4.2.2 --- Relative expression levels of Raldh2 --- p.50 / Chapter 3.4.2.3 --- Relative expression levels of Raldh3 --- p.51 / Chapter 3.4.3 --- Comparison of the in vitro RA synthesizing activity of Raldh 1 Raldh2 and Raldh3 enzymes between embryos of diabetic and non-diabetic mice at different developmental stages --- p.52 / Chapter 3.5 --- Discussion --- p.55 / Chapter Chapter 4: --- Effect of Hyperglycemia on Retinoic Acid Synthesis / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.2 --- Experimental Design --- p.61 / Chapter 4.3 --- Materials and Methods --- p.64 / Chapter 4.3.1 --- Phlorizin treatment --- p.64 / Chapter 4.3.2 --- Whole rat embryo culture --- p.64 / Chapter 4.3.3 --- Preparation of rat serum --- p.65 / Chapter 4.3.4 --- In situ hybridization --- p.66 / Chapter 4.3.5 --- Real-time quantitative RT-PCR --- p.66 / Chapter 4.3.6 --- In vitro RA synthesizing reaction --- p.68 / Chapter 4.3.7 --- Statistical analyses --- p.68 / Chapter 4.4 --- Results --- p.70 / Chapter 4.4.1 --- "Comparison of the relative expression level of Raldh 1, Raldh2 and Raldh3 between embryos of diabetic and non-diabetic mice injected with phlorizin or suspension vehicle as control" --- p.70 / Chapter 4.4.2 --- Comparison of the in vitro RA synthesizing activity of different isoforms of Raldh enzymes between embryos of diabetic and non-diabetic mice injected with phlorizin or suspension vehicle as control --- p.73 / Chapter 4.4.3 --- In situ expression pattern of Raldh2 in rat embryos cultured in medium containing varying concentrations of D-glucose --- p.77 / Chapter 4.4.4 --- Relative expression levels of Raldh2 in rat embryos cultured in medium supplemented with varying concentrations of D-glucose --- p.78 / Chapter 4.4.5 --- In vitro RA synthesizing activity ofRaldh2 in rat embryos cultured in medium supplemented with varying concentrations of D-glucose --- p.79 / Chapter 4.5 --- Discussion : --- p.82 / Chapter Chapter 5: --- In Vitro Supplementation with RA Rescued Rat Embryos from Hyperglycemia-induced Congenital Malformations / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Experimental Design --- p.88 / Chapter 5.3 --- Materials and Methods --- p.89 / Chapter 5.3.1 --- Preparation of RA --- p.89 / Chapter 5.3.2 --- Supplementation of RA to rat embryos in culture --- p.89 / Chapter 5.3.3 --- Morphological scoring system --- p.90 / Chapter 5.3.4 --- Statistical analyses --- p.90 / Chapter 5.4 --- Results --- p.92 / Chapter 5.4.1 --- Supplementation with RA rescued embryos from hyperglyce- miainduced malformations --- p.92 / Chapter 5.5 --- Discussion --- p.101 / Chapter Chapter 6: --- Conclusion and Future Perspectives / Chapter 6.1 --- Conclusion and Future Perspectives --- p.106 / References --- p.111

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327451
Date January 2011
ContributorsChan, Wing Lung., Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvii, 130 leaves, [5] leave of plates : ill. (chiefly col.) ; 30 cm.
RightsUse 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/)

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