Lee, Man Yuen. / "March 2008." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 103-128). / Abstracts in English and Chinese. / Title Page --- p.i / Acknowledgements --- p.ii / Table of Content --- p.iii / List of Figures --- p.vii / List of Graphs --- p.viii / List of Tables --- p.x / Abbreviations --- p.xii / Abstract --- p.xiii / Abstract (Chinese) --- p.xv / 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 --- p.5 / Chapter 1.2 --- Diabetic pregnancy --- p.6 / Chapter 1.3 --- Etiology of diabetes-induced malformations --- p.9 / Chapter 1.3.1 --- Hyperglycaemia --- p.10 / Chapter 1.3.2 --- Hyperketonaemia and somatomedin inhibitors --- p.10 / Chapter 1.3.3 --- Oxidative stress --- p.11 / Chapter 1.3.4 --- Deficiency of myo-inositol and arachidonic acid --- p.12 / Chapter 1.4 --- Vitamin A --- p.13 / Chapter 1.5 --- Retinoic acid --- p.14 / Chapter 1.5.1 --- RA signaling on embryo development --- p.15 / Chapter 1.5.2 --- RA teratogenicity --- p.15 / Chapter 1.5.3 --- RA regulation --- p.17 / Chapter 1.6 --- RA and maternal diabetes-induced caudal regression share similar pathogenic mechanisms --- p.19 / Chapter 1.7 --- Strategy of the thesis --- p.21 / Chapter Chapter 2: --- General Materials and Methods / Chapter 2.1 --- Animal --- p.25 / Chapter 2.2 --- Induction of diabetes --- p.25 / Chapter 2.3 --- Preparation of retinoic acid for mouse injection --- p.26 / Chapter 2.4 --- RA responsive cell line --- p.26 / Chapter 2.4.1 --- Cell culture --- p.27 / Chapter 2.4.2 --- Seeding and adding sample to 96-well plate --- p.28 / Chapter 2.4.3 --- Staining of cells --- p.28 / Chapter 2.5 --- Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) --- p.29 / Chapter 2.5.1 --- Collection and storage of tissues --- p.29 / Chapter 2.5.2 --- Total RNA extraction --- p.29 / Chapter 2.5.3 --- Reverse transcription --- p.30 / Chapter 2.5.4 --- Polymerase chain reaction --- p.30 / Chapter 2.5.5 --- Preparation of cDNA standard --- p.31 / Chapter 2.5.6 --- Mini-scale preparation of plasmid DNA --- p.32 / Chapter Chapter 3: --- Effects of Maternal Diabetes on RA Catabolism in the Tail Bud Region / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.2 --- Experimental design --- p.37 / Chapter 3.3 --- Materials and methods --- p.38 / Chapter 3.3.1 --- Preparation of RA stock solution for cell culture --- p.38 / Chapter 3.3.2 --- Preparation of RA standard solutions --- p.38 / Chapter 3.3.3 --- Characterization of RA responsive cell line --- p.39 / Chapter 3.3.3.1 --- Determining optimal culture time for maximum response --- p.39 / Chapter 3.3.3.2 --- Testing toxicity of DMSO --- p.39 / Chapter 3.3.3.3 --- Detection of β-galactosidase activity by β_gal Assay Kit --- p.40 / Chapter 3.3.4 --- In vitro assay of enzymatic degradation of RA --- p.41 / Chapter 3.3.4.1 --- Testing toxicity of enzyme cofactor-reducing agent --- p.41 / Chapter 3.3.4.2 --- Collection of tail bud --- p.42 / Chapter 3.3.4.3 --- In vitro enzymatic reaction --- p.43 / Chapter 3.3.5 --- In vivo assay of enzymatic degradation of RA --- p.44 / Chapter 3.3.5.1 --- Determining the optimal time for maximum release of RA from the tail bud into the medium --- p.44 / Chapter 3.3.5.2 --- Monitoring of RA remained in the tail bud after injection of exogenous RA --- p.45 / Chapter 3.3.6 --- Statistical analysis --- p.45 / Chapter 3.4 --- Results --- p.46 / Chapter 3.4.1 --- Optimization of RA responsive cell line --- p.46 / Chapter 3.4.1.1 --- Effect of incubation time with RA on β-galactosidase expression level --- p.46 / Chapter 3.4.1.2 --- Toxicity of RA and DMSO --- p.47 / Chapter 3.4.1.3 --- Comparison of X-gal staining assay and β_gal Assay Kit for detection of β-galactosidase expression --- p.48 / Chapter 3.4.2 --- In vitro assay of RA catabolic activity in the tail bud --- p.49 / Chapter 3.4.2.1 --- Toxicity of enzyme cofactor-reducing agent --- p.49 / Chapter 3.4.2.2 --- RA catabolic activity in lysed and intact tail buds --- p.50 / Chapter 3.4.2.3 --- Effect of enzyme cofactor and inhibitor --- p.50 / Chapter 3.4.2.4 --- Comparsion of in vitro RA catabolic activity of diabetic and non-diabetic groups --- p.51 / Chapter 3.4.3 --- In vivo assay of RA catabolic activity in the tail bud --- p.52 / Chapter 3.4.3.1 --- Optimal time for maximum release of RA from the tail bud into the medium --- p.53 / Chapter 3.4.3.2 --- Comparison of RA remained in the tail bud of embryos of diabetic and non-diabetic mice --- p.53 / Chapter 3.5 --- Discussion --- p.55 / Chapter Chapter 4: --- Analysis of Cyp26 Expression in the Tail Bud Region / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Experimental design --- p.63 / Chapter 4.3 --- Materials and methods --- p.64 / Chapter 4.3.1 --- Sample collection --- p.64 / Chapter 4.3.2 --- Real-time quantitative RT-PCR --- p.64 / Chapter 4.3.3 --- Statistical analysis --- p.66 / Chapter 4.4 --- Results --- p.67 / Chapter 4.4.1 --- "Relative expression levels of Cyp26al, Cyp26bl and Cyp26cl" --- p.67 / Chapter 4.4.2 --- Molecular changes in Cyp26al in tail bud region after maternal RA treatment --- p.68 / Chapter 4.5 --- Discussion --- p.72 / Chapter Chapter 5: --- Comparison of Cyp26al Heterozygous and Wild-type Embryos in Diabetic and Non-diabetic Pregnancies / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.2 --- Experimental design --- p.79 / Chapter 5.3 --- Materials and methods --- p.81 / Chapter 5.3.1 --- Animal --- p.81 / Chapter 5.3.2 --- DNA genotyping --- p.81 / Chapter 5.3.3 --- Measurement of RA remained in the tail bud after RA treatment --- p.82 / Chapter 5.3.4 --- Analysis of extent of caudal regression --- p.83 / Chapter 5.3.5 --- Real-time quantitative RT-PCR --- p.83 / Chapter 5.3.6 --- Statistical analysis --- p.84 / Chapter 5.4 --- Results --- p.85 / Chapter 5.4.1 --- Comparsion of pregnancy outcome of mating with ICR or Cyp26al+/- males --- p.85 / Chapter 5.4.2 --- Expression levels of Cyp26al determined by real-time quantitative RT-PCR --- p.85 / Chapter 5.4.3 --- Determination of RA catabolic activity --- p.86 / Chapter 5.4.4 --- Extent of caudal regression --- p.90 / Chapter 5.5 --- Discussion --- p.93 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.96 / References --- p.102 / Figures / Graphs
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326274 |
| Date | January 2008 |
| Contributors | Lee, Man Yuen., Chinese University of Hong Kong Graduate School. Division of Obstetrics and Gynaecology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xv, 128 leaves : col. ill. ; 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/) |
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