by Yuen-Kam Tsoi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 160-173). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / List of figures --- p.vii / List of tables --- p.x / Abbreviations --- p.xi / Chapter Chapter 1 --- Purification and characterization of the chymotrypsin inhibitor (MCCI) from the seeds of Momordica cochinchinensis / Chapter 1.1 --- Introduction l / Chapter 1.1.1 --- Classification of protease inhibitor --- p.2 / Chapter 1.1.2 --- Therapeutic potential of protease inhibitors --- p.4 / Chapter 1.2 --- Rationale of the present study --- p.6 / Chapter 1.3 --- Materials and methods / Chapter 1.3.1 --- Materials --- p.9 / Chapter 1.3.2 --- Preparation of chymotrypsin-Sepharose 4B affinity column --- p.10 / Chapter 1.3.3 --- Protein extraction --- p.11 / Chapter 1.3.4 --- Chymotrypsin-Sepharose 4B affinity chromatography --- p.12 / Chapter 1.3.5 --- Reversed phase high pressure liquid chromatography --- p.12 / Chapter 1.3.6 --- Assays for protease inhibitory activities --- p.14 / Chapter 1.3.6.1 --- Assay for chymotrypsin activity --- p.15 / Chapter 1.3.6.2 --- Assay for trypsin activity --- p.15 / Chapter 1.3.6.3 --- Assay for elastase activity --- p.16 / Chapter 1.3.6.4 --- Assay for subtilisin activity --- p.16 / Chapter 1.3.7 --- Determination of protein concentration --- p.17 / Chapter 1.3.8 --- Titration of chymotrypsin --- p.17 / Chapter 1.3.9 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.18 / Chapter 1.3.10 --- Determination of molecular weight by mass spectrometry --- p.19 / Chapter 1.3.11 --- Partial amino acid sequencing --- p.20 / Chapter 1.3.12 --- Effects of chymotrypsin on MCCI --- p.20 / Chapter 1.3.13 --- Stability assay --- p.21 / Chapter 1.4 --- Results --- p.21 / Chapter 1.4.1 --- Isolation of MCCI from the seeds of Momordica cochinchinensis --- p.21 / Chapter 1.4.2 --- N-terminal amino acid sequencing --- p.27 / Chapter 1.4.3 --- Determination of molecular weight --- p.31 / Chapter 1.4.4 --- Inhibitory activity of MCCI towards different proteases --- p.33 / Chapter 1.4.5 --- Effects of chymotrypsin on MCCI --- p.37 / Chapter 1.4.6 --- Stability of MCCI on heating and at different pH --- p.37 / Chapter 1.5 --- Discussion --- p.42 / Chapter Chapter 2 --- Immunomodulatory effect of MCCI / Chapter 2.1 --- Introduction of the immune system and protease inhibitors --- p.51 / Chapter 2.2 --- Rationale of the present study --- p.55 / Chapter 2.3 --- Materials and methods --- p.56 / Chapter 2.3.1 --- Materials --- p.56 / Chapter 2.3.2 --- Isolation of different types of immune cells --- p.57 / Chapter 2.3.3 --- Determination of cell proliferation --- p.60 / Chapter 2.3.4 --- Determination of H2O2 formation --- p.61 / Chapter 2.3.5 --- Assay of interleukin-2 --- p.61 / Chapter 2.3.6 --- Determination of cell viability --- p.62 / Chapter 2.4 --- Results --- p.63 / Chapter 2.4.1 --- Murine splenocytes --- p.63 / Chapter 2.4.1.1 --- In vitro effect of MCCI on the proliferation of murine splenocytes --- p.63 / Chapter 2.4.1.2 --- Effect of MCCI on cytokine production --- p.63 / Chapter 2.4.2 --- Murine lymphocytes --- p.66 / Chapter 2.4.2.1 --- In vitro effect of MCCI on the proliferation of lymphocytes --- p.66 / Chapter 2.4.2.2 --- Effect of MCCI on cytokine production --- p.66 / Chapter 2.4.3 --- Murine bone marrow cells --- p.69 / Chapter 2.4.3.1 --- Effect of MCCI on the growth of murine bone marrow cells --- p.69 / Chapter 2.4.4 --- Murine neutrophills --- p.69 / Chapter 2.4.4.1 --- Effect of MCCI on H2O2 formation --- p.69 / Chapter 2.4.5 --- Murine macrophages --- p.71 / Chapter 2.4.5.1 --- Effect of MCCI on the growth of macrophages --- p.71 / Chapter 2.4.5.2 --- Effect of external ATP on the growth of macrophages --- p.71 / Chapter 2.4.5.3 --- Effect of ATP on the growth of macrophages pre-treated with MCCI --- p.76 / Chapter 2.4.5.4 --- Effect of MCCI on the growth of macrophages pre-treated with ATP --- p.76 / Chapter 2.4.5.5 --- Effect of MCCI on H201production --- p.79 / Chapter 2.5 --- Discussion --- p.82 / Chapter Chapter 3 --- Anti-oxidative effect of MCCI in primary rat hepatocytes culture / Chapter 3.1 --- Introduction --- p.91 / Chapter 3.1.1 --- Liver disease and protease inhibitors --- p.91 / Chapter 3.1.2 --- Primary rat hepatocyte as a pharmacological model --- p.93 / Chapter 3.1.3 --- tert-Butyl hydroperoxide as an oxidative stress inducer --- p.94 / Chapter 3.1.4 --- Endogenous antioxidant enzymes against ROS --- p.96 / Chapter 3.2 --- Rationale of the present study --- p.99 / Chapter 3.3 --- Materials and methods --- p.101 / Chapter 3.3.1 --- Materials --- p.101 / Chapter 3.3.2 --- Isolation of primary rat hepatocytes --- p.101 / Chapter 3.3.2.1 --- Liver perfusion --- p.101 / Chapter 3.3.2.2 --- Preparation of collagen pre-coated culture plates --- p.103 / Chapter 3.3.2.3 --- Hepatocytes culturing --- p.103 / Chapter 3.3.3 --- Drug treatment and oxidative stress induction --- p.104 / Chapter 3.3.4 --- Cytotoxicity assessment --- p.105 / Chapter 3.3.5 --- Cellular GSH content determination --- p.105 / Chapter 3.3.6 --- Protein determination by Lowry's method --- p.106 / Chapter 3.3.7 --- Medium MDA determination --- p.106 / Chapter 3.3.8 --- Medium GSSG determination --- p.107 / Chapter 3.3.9 --- Antioxidant enzymes measurement --- p.108 / Chapter 3.3.9.1 --- Catalase measurement --- p.108 / Chapter 3.3.9.2 --- SOD measurement --- p.109 / Chapter 3.3.9.3 --- GST measurement --- p.109 / Chapter 3.3.9.4 --- GR measurement --- p.110 / Chapter 3.3.10 --- Statistical analysis --- p.110 / Chapter 3.4 --- Results --- p.111 / Chapter 3.4.1 --- Cytotoxicity of MCCI on rat hepatocytes --- p.111 / Chapter 3.4.2 --- Effect of tBHP and MCCI on hepatocytes viability --- p.111 / Chapter 3.4.3 --- Effects of tBHP and MCCI on hepatocytes GSH and GSSG content --- p.117 / Chapter 3.4.4 --- Effect of MCCI on lipid peroxidation of hepatocytes --- p.121 / Chapter 3.4.5 --- Effect of MCCI on antioxidant enzymes activities --- p.121 / Chapter 3.4.6 --- Comparison with typical antioxidants --- p.125 / Chapter 3.5 --- Discussion --- p.127 / Chapter Chapter 4 --- Cytotoxicity of MCCI on tumor cell lines / Chapter 4.1 --- Introduction --- p.134 / Chapter 4.1.1 --- Relationship between protease inhibitors and cancer --- p.134 / Chapter 4.1.2 --- Cell cycle and apoptosis --- p.137 / Chapter 4.2 --- Rationale of the present study --- p.140 / Chapter 4.3 --- Materials and methods --- p.141 / Chapter 4.3.1 --- Materials --- p.141 / Chapter 4.3.2 --- Cell culture --- p.141 / Chapter 4.3.3 --- MTT assay --- p.142 / Chapter 4.3.4 --- Cell cycle analysis --- p.142 / Chapter 4.3.5 --- DNA fragmentation --- p.143 / Chapter 4.4 --- Results --- p.136 / Chapter 4.4.1 --- Cytotoxicity of MCCI --- p.144 / Chapter 4.4.2 --- Cell cycle and apoptosis analysis --- p.147 / Chapter 4.5 --- Discussion --- p.152 / Conclusion and future perspectives --- p.157 / References --- p.160
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324408 |
| Date | January 2003 |
| Contributors | Tsoi, Yuen-Kam., 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, xiv, 173 leaves : ill. ; 30 cm. |
| Coverage | China |
| 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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