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University of Southampton Research Repository Eprints Soton University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk UNIVERSITY OF SOUTHAMPTON REGULATION OF THE SYNTHESIS OF TISSUE INHIBITORS OF METALLOPROTEINASE-1 AND –2 BY HEPATIC AND PANCREATIC STELLATE CELLS Dr Peter Raymond McCrudden B.Sc.(Hons) MBBS FRCP Submitted for the degree of MD Faculty of Medicine, Health and Biological Sciences June 2010 For Ute, Nicolas, Katia and India 2 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF MEDICINE, HEALTH AND LIFE SCIENCES (MHLS) SCHOOL OF MEDICINE DIVISION OF INFECTION, INFLAMMATION AND IMMUNITY Doctor of Medicine REGULATION OF THE SYNTHESIS OF TISSUE INHIBITORS OF METALLOPROTEINASE-1 AND –2 BY HEPATIC AND PANCREATIC STELLATE By Dr Peter Raymond McCrudden Hepatic stellate cells (HSC) play a central role in fibrosis development by production of extracellular matrix and also by secretion of matrix metalloproteinases (MMPs) and Tissue inhibitors of metalloproteinases (TIMPs) including TIMP-2 and MMP-2. TIMP-2 has been shown to interact with Gelatinase A in conjunction with MT1-MMP (MMP-14). TIMP-2 has been traditionally considered to be constitutively expressed. There is some evidence that TIMP-2 expression is slightly enhanced in fibrotic disease and activation in tissue culture. Little is known in terms of TIMP-2 expression in the recently described pancreatic stellate cells (PSC). HSC were cultured on plastic having been isolated from rat and human liver resections. After culture on plastic northern analysis was performed for TIMP-2 mRNA expression. TIMP-2 promoter activity was examined in rat pancreatic stellate cells (PSC) and rat hepatic stellate cells. Early work led to subcloning the promoter into a different vector though subsequent promoter studies were unsuccessful. In vivo work in immunohistochemistry studies suggest there is increased TIMP-2 expression in evaluation of rat pancreas and liver in addition to human liver and pancreas specimens. By ribonuclease protection assay TIMP-2 was noted to be upregulated in human fibrotic liver compared to normal human liver tissue. In conclusion there is some evidence that TIMP-2 regulation may be altered in fibrotic liver states as well as in pancreatic inflammatory disease. 3 LIST OF CONTENTS ABSTRACT 3 LIST OF CONTENTS 4 LIST OF FIGURES 10 LIST OF TABLES 13 LIST OF TABLES 13 ACKNOWLEDGEMENTS 14 ACKNOWLEDGEMENTS 14 DECLARATION 15 LIST OF ABBREVIATIONS 16 INTRODUCTION 19 1.1 Background to liver disease and hepatic fibrosis 20 1.2 Anatomy of the liver 20 1.3 Structure of the normal liver sinusoid 23 1.4 The extracellular matrix of normal and diseased liver 24 1.5 Identification of Hepatic Stellate Cells (HSCs) as the primary source of Extracellular Matrix 30 1.6 Cellular sources of Extracellular Matrix (ECM) 32 1.7 Cellular features of Quiescent HSCs 33 1.8 Cellular features of HSC activation 34 1.8.1 Mechanism of HSC activation 36 1.8.2 Perpetuation: the Paracrine and autocrine driven cytokine activity and ECM remodeling that sustain the activated phenotype 38 1.8.3 mRNA stability 40 1.8.3.1 Regulation of mRNA degradation 40 1.8.4 HSC and Matrix degradation 41 1.8.5 Phenotypic characterization of quiescent and activated HSCs 43 1.9 ECM degradation: role in cirrhosis 45 1.10 The Tissue Inhibitors of Metalloproteinase (TIMP) 48 4 1.11 Evidence for change in matrix degradation during liver fibrosis 50 1.12 Evidence for TIMP expression in progressive liver fibrosis 51 1.13 Resolution of fibrosis 53 1.14 Control of apoptosis in the liver 56 1.15 Pancreas Fibrosis and Pancreatic Stellate Cells 57 1.16 Clinical Setting of Pancreatic Disease 59 1.17 Properties of PSCs 60 1.18 Mediators of PSC activation 62 1.19 PSCs and pancreatic inflammation 64 1.20 Inflammation and PSC modulation of ECM 66 1.21 PSCs and pancreatic cancer 67 1.22 Comparison of PSCs and HSCs 69 1.23 Hypoxia, Oxygen and genes in health and disease: an introduction 71 1.23.1 HIF-1 activation by Hypoxia 72 1.23.3 The sensor control 73 1.23.4 Hypoxia studies in the liver 73 1.24 Inhibition of Tissue inhibitors of Metalloproteinases 74 1.25 Hypothesis 75 1.26 AIMS 76 CHAPTER 2 77 2.1 Materials 78 2.1.1 General Methods 78 2.1.2 Immunohistochemistry 78 2.1.3 Method 1: Preparation of slides 80 2.1.4 Method 2: Antigen Retrieval -Proteolytic Enzyme -Pronase Pre-treatment 81 2.1.5 Method 3: Antigen Retrieval - heat mediated – microwave pre-treatment using citrate or EDTA buffer 82 2.1.6 Method 4: Antigen Retrieval -Heat-mediated -Pressure Cooker Pre-treatment 83 2.1.7 Method 5: Demonstration of Enzyme Peroxidase using Biomed Liquid DAB Substrate Kit 84 2.1.8 Method 6: Streptavidin-Biotin Peroxidase Complex (StABCPx) immunostaining technique for fixed, paraffin embedded sections using monoclonal or polyclonal antibodies 85 2.2 Isolation and Culture of Hepatic Cells 87 5 2.2.1 Method of Isolation of Rat Hepatic Stellate Cell 87 2.2.2 Preparation of Human Hepatic Stellate Cells 89 2.2.3 Assessment of Cell Number and Viability 90 2.2.4 Cell Culture 90 2.2.5 Characterization of Hepatic Stellate Cells 90 2.2.6 Cell Purity 91 2.2.7 Pancreatic Stellate Cells Isolation 91 2.2.8 Trypsinization and Passaging Of Pancreatic Stellate Cells 92 2.2.9 Preparation of conditioned media 93 2.2.10 Trypsinization of adherent cells 93 2.3 Extraction, Purification and Analysis of RNA 94 2.3.1 Extraction of RNA 94 2.3.2 General precautions for working with RNA 94 2.3.3 Preparation of GIT lysate 95 2.3.4 RNA extraction by the acid phenol method 95 2.3.5 Electrophoresis of RNA 96 2.3.6 Northern Blotting 97 2.3.7 Hybridisation of Northern Blots with Radiolabeled cDNA Probes 97 2.3.8 Pre-Hybridisation of Northern Blots 98 2.3.9 Radiolabeling Of cDNA Probes By Random Priming 98 2.3.10 Stringency washing of Northern blots 99 2.4 Reverse Transcription- Polymerase Chain Reaction (RT-PCR) of Messenger RNA Sequences 99 2.4.1 Synthesis of Double Stranded DNA Copy 100 2.4.2 Polymerase Chain Reaction 100 2.5 Ribonuclease Protection Assays 102 2.5.1 Preparation of Radiolabeled Antisense Riboprobe 105 2.5.2 Phenol chloroform extraction of cDNA templates 105 2.5.3 In Vitro Transcription of Radiolabeled Antisense Riboprobe 106 2.5.4 Protocol for Ribonuclease Protection Assay 106 2.5.5 Sample Preparation 107 6 2.5.6 Resolution of Protected Fragments on Polyacrylamide Urea Gels 107 2.6 Amplification, Purification and Analysis of Plasmid DNA 109 2.6.1 Purification of Plasmid DNA 110 2.6.2 Restriction Enzyme Analysis of Plasmid DNA 110 2.6.3 Preparation of Plasmid DNA for cDNA Probes, Riboprobes, Transfections, and Nuclear Run Ons 111 2.6.4 Amplification, Purification and Analysis of Plasmid DNA 111 2.6.5 Production of competent cells (DH5 α E. coli) 111 2.6.6 Transformation and Amplification of competent Escherichia coli (E coli) with Plasmid DNA 112 2.6.7 Bacterial Propagation 113 2.6.8 Production of Bacterial Stock 114 2.6.9 Purification of Plasmid DNA 114 2.6.10 Restriction Enzyme analysis of Plasmid DNA 116 2.6.11 Purification of DNA or PCR products from Agarose Gels 116 2.7.1 Transfection techniques: Introduction 118 2.7.2 Luciferase Reporter Assay System 119 2.7.3 Transfection 122 2.7.4 The Chloramphenicol Acetyl Transferase (CAT) Transfection Assay 122 2.8 Erase-a-Base 124 2.8.1 Erase-a-Base system for restriction digestion of plasmid DNA 126 2.8.1.1 Protection of 5`-Protruding Ends with α-Phosphorothioate dNTP Mix 126 2.8.2 Exonuclease III deletion, ligation and transformation 126 2.9 Sequencing Methods 127 2.9.1 Chain Termination Sequencing 127 2.9.2 Sequencing Reactions 129 2.9.3 Termination Reaction 129 2.9.4 Labeling Reaction 130 2.9.5 Termination Reaction 130 2.9.6 Acrylamide Gel 130 2.10 Culture of cells in hypoxic condition 131 2.11 Statistical Analysis 132 7 CHAPTER 3 133 3.1 INTRODUCTION 134 3.2 Methods 136 3.2.1 Ethics Approval 136 3.2.2 Selection of cases - Pancreas 136 3.2.3 Selection of cases – Liver 139 3.3 Results of Immunohistochemistry 144 3.4 Discussion 157 3.4.1 Pancreas 157 3.4.2 Liver 157 CHAPTER 4 160 4.1 Introduction 161 4.1.1 Northern Blotting for the detection of α SMA, Procollagen-1, Gelatinase A, TIMPs -1 & -2 in Activated rat pancreatic stellate cells (PSC) 161 4.1.2 Ribonuclease Protection Assay in human livers for expression of TIMP-2 162 4.2 Methods 164 4.2.1 Methods RNA expression are described in the Methods chapter section 2.27 & 2.3 165 4.2.2 Methods RPAs are described in the Methods chapter 2 section 2.5 165 4.3.1 Results 166 4.3.2 Results 172 4.3.3 Results 173 4.4 Discussion 174 CHAPTER 5 177 5.1 Introduction 178 5.2 Methods 186 5.3 Results 187 5.3.1 Early Transfection work with the luciferase reporter assay 187 5.4 Discussion on Early Transfection 190 5.5 Subcloning plasmid DNA into another plasmid vector 191 5.5.1 Aim: Isolation of the TIMP-2 full promoter (2600bp) insert in the pGL3 vector
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