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 Faculty of Medicine, Health and Biological Sciences Pancreatic Research Group An investigation into soluble growth factors of TIMP-1, IGF-1 and Insulin on Pancreatic stellate cell survival Dr Manish Patel DM Thesis February 2014 University of Southampton An investigation into soluble growth factors of TIMP-1, IGF-1 and Insulin on Pancreatic stellate cell survival Faculty of Medicine, Health and Biological Sciences During pancreatic injury, the pancreatic stellate cell(PSC) become activated to a myofibroblast-like phenotype, proliferate and are known to be the major source of matrix which characterise pancreatic fibrosis in chronic pancreatitis and pancreatic cancer. Activated PSC also express matrix degrading metalloproteinases(MMPs) and their tissue inhibitors(TIMPs). Previous work has demonstrated that during spontaneous recovery from experimental liver fibrosis after 4 weeks of carbon tetrachloride injections, there is a fall in the expression of TIMP-1, a loss of the hepatic stellate cells (HSCs) by apoptosis, and an increase in liver collagenolytic activity with the return of the liver to a near normal histology. This correlation between PSC survival and expression of TIMP-1 in vivo highlighted potential roles for TIMP-1, IGF-1 and insulin in PSC survival and apoptosis which was the subject of this study. The initial aim this thesis was to establish and characterise human PSC in culture. This was achieved and cells were studied between passage 1 and 4 in order to determine the effect of TIMP-1, IGF-1 and insulin on PSC survival. These data suggest that TIMP-1, showed a dose dependent anti apoptotic effect in activated rat and human PSCs, with no effect on proliferation. Additionally, successful silencing of TIMP-1 at the protein and mRNA level using electroporation was achieved, without significantly effecting PSC cytotoxicity. Furthermore, electroporation with TIMP-1 significantly increased the level of apoptosis compared with control, and this effect was attenuated by the addition of recombinant TIMP-1. IGF-1 and insulin receptors were expressed in vitro, in activated rat PSCs. Immunohistochemistry demonstrated weak IGF-1R positive staining within areas of fibrosis in pancreatic resection specimens, which co-localised to cells which also stained positive for -SMA a marker of activated PSCs. IGF-1 induced an increase in PSC numbers by promoting survival as well as increasing proliferation. On serum withdrawal IGF-1 is secreted by activated PSC. Insulin promotes survival but has no significant effect on proliferation. In conclusion these data have identified TIMP-1, IGF-1 and insulin as mediators of stellate cell survival via inhibition of apoptosis. Furthermore, a successful technique has been identified to knockdown siRNA in PSC to allow further mechanistic studies to be performed. Contents i) Abstract……………………………………………………………………………………………………………..……2 ii) Contents………………………………………..…………………………………….…………………………..……..3 iii) Table of figures………………………………………………………………………………………………………10 iv) Declaration of Authorship …………………………………………………..………………………………..13 v) Acknowledgments…………….………..…………………………………………………………………………14 1 Chapter 1 ............................................................................................................................... 15 1.1 Normal Pancreatic anatomy ......................................................................................... 15 1.1.1 Macroscopic anatomy ........................................................................................... 15 1.1.2 Microscopic anatomy ............................................................................................ 17 1.1.3 Fine structure of ducts .......................................................................................... 19 1.1.4 Insulo-acinar portal system ................................................................................... 20 1.2 Chronic pancreatitis ...................................................................................................... 21 1.2.1 Aetiology of chronic pancreatitis .......................................................................... 22 1.2.2 Toxic and metabolic factors. ................................................................................. 22 1.2.3 Idiopathic chronic pancreatitis. ............................................................................ 23 1.2.4 Genetic predisposition. ......................................................................................... 23 1.2.5 Autoimmune pancreatitis ..................................................................................... 24 1.2.6 Recurrent and severe pancreatitis ........................................................................ 25 1.2.7 Obstructive chronic pancreatitis ........................................................................... 25 3 1.2.8 Diagnosis of Chronic Pancreatitis .......................................................................... 25 1.3 Complications ................................................................................................................ 26 1.4 Treatment of chronic pancreatitis ................................................................................ 27 1.5 Pancreatic fibrosis and the role of pancreatic stellate cells ......................................... 27 1.5.1 Quiescent Pancreatic Stellate cells ....................................................................... 28 1.5.2 Similarities between HSC and PSC ........................................................................ 28 1.5.3 Isolation of pancreatic stellate cells and in vitro activation ................................. 29 1.5.4 Mechanisms of PSC activation .............................................................................. 30 1.5.5 Autocrine loops and intracellular signal mediation in PSC ................................... 31 1.5.6 Signalling Pathways ............................................................................................... 32 1.5.7 High Glucose and pancreatic stellate cells ............................................................ 33 1.6 Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases .................... 34 1.6.1 MMPs and TIMPs in pancreatic disease................................................................ 35 1.7 The effects of lipopolysaccharide on stellate cells ....................................................... 37 1.8 Apoptosis in the resolution of fibrosis .......................................................................... 39 1.9 Potential therapeutic approaches for pancreatic fibrosis ............................................ 45 1.10 Hypothesis and Aims ..................................................................................................... 45 1.10.1 Hypothesis ............................................................................................................. 46 1.10.2 Aims of the study: ................................................................................................. 46 2 Chapter 2 ............................................................................................................................... 47 4 2.1 Methods ........................................................................................................................ 47 2.1.1 Cell Culture ............................................................................................................ 47 2.1.2 Isolation of human PSCs ........................................................................................ 48 2.1.3 Culture of pancreatic stellate cells ........................................................................ 48 2.1.4 Trypsinisation of pancreatic stellate cell ............................................................... 49 2.2 Stimulation and quantification of apoptosis................................................................. 49 2.2.1 Detection of apoptosis using acridine orange staining to look at nuclear morphology. .......................................................................................................................... 50 2.2.2 Caspase-3 Activity assay........................................................................................ 50 2.2.3 Quantification of apoptosis using Apo-ONE Homogenous Caspase 3/7 Assay in siRNA treated PSC ................................................................................................................. 51 2.3 Quantification of DNA synthesis ................................................................................... 52 2.4 Protein methods ..........................................................................................................