Function of ion transport proteins in pancreatic stellate cells (PSCs)

Das Pankreaskarzinom ist durch ein fibrotisches und hypoxisches Tumormikromilieu gekennzeichnet. Daran sind vor allen pankreatische Sternzellen beteiligt. Sternzellen und Krebszellen aktivieren sich gegenseitig parakrin. Das führt auch zur Stimulation der Migration von Sternzellen, was Voraussetzung...

Author: Nielsen, Nikolaj
Further contributors: Schwab, Albrecht (Thesis advisor)
Division/Institute:FB 13: Biologie
FB 05: Medizinische Fakultät
Document types:Doctoral thesis
Media types:Text
Publication date:2015
Date of publication on miami:25.06.2015
Modification date:27.07.2015
Edition statement:[Electronic ed.]
Subjects:Pankreatische Sternzellen; TRP Kanäle; Migration; Tumormilieu; Mikromilieu- sensor/Modifikator/Effektor PSC; TRP channels; migration; tumour microenvironment; microenvironmental sensors/modifiers/effectors
DDC Subject:570: Biowissenschaften; Biologie
License:InC 1.0
Language:English
Format:PDF document
URN:urn:nbn:de:hbz:6-29229486062
Permalink:http://nbn-resolving.de/urn:nbn:de:hbz:6-29229486062
Digital documents:diss_nielsen.pdf
Table of contents:
  • DECLARATION AND CERTIFICATION OF ORIGINALITY ................................................................................... i
  • PREFACE ........................................................................................................................................................................... ii
  • ACKNOWLEDGEMENTS ............................................................................................................................................. iii
  • CURRICULUM VITAE ................................................................................................................................................... iv
  • CONTRIBUTIONS BY THE AUTHOR ....................................................................................................................... vi
  • OVERALL AIM OF THE IONTRAC PROJECT .......................................................................................................... ix
  • SUMMARY ........................................................................................................................................................................ x
  • GERMAN RESÚME......................................................................................................................................................... xi
  • TABLE OF CONTENTS ................................................................................................................................................... I
  • ABBREVIATIONS ........................................................................................................................................................... V
  • INTRODUCTION
  • 1.1 Anatomy and Function of the Pancreas .........................................................................1
  • 1.2 Pancreatic Ductal Adenocarcinoma ...............................................................................3
  • 1.2.1 Clinical Manifestation and Risk Factors.................................................................................. 3
  • 1.2.2 Characterisation and Molecular Pathogenesis of PDAC ........................................................ 4
  • 1.3 Pancreatic Stellate Cells – Key Players in PDAC Progression and Metastasis .............6
  • 1.3.1 The History and Characterization of Pancreatic Stellate Cells ............................................... 7
  • 1.3.2 Pancreatic Stellate Cell Activation.......................................................................................... 8
  • 1.3.3 Pancreatic Stellate Cells in the (PDAC) Microenvironment .................................................10
  • 1.3.3.1 Hypoxia – A Major Feature in PDAC and PSC Activation ............................................................. 11
  • 1.3.3.2 PSCs Mutual Signalling with PDAC Cells ...................................................................................... 12
  • 1.3.4 Autocrine Stimulation in PSCs ..............................................................................................14
  • 1.4 Cell Migration ................................................................................................................ 15
  • 1.4.1 Basic Principles of Cell Migration .........................................................................................15
  • 1.4.2 Calcium-dependent Signalling in Cell Migration ..................................................................17
  • 1.5 TRP Channels in Migration and as Microenvironmental Sensors, Modifiers and
  • Effectors ............................................................................................................................... 19
  • 1.5.1 TRP Channel Family ..............................................................................................................19
  • 1.5.1.1 TRP Channel Function .................................................................................................................. 19
  • 1.5.1.2 TRP Channels in Cell Migration and Interaction Partners ............................................................ 20
  • 1.5.2 TRP Channels as Sensors and Effectors of the Tumour Microenvironment ........................23
  • 1.5.2.1 TRP Channels as Mechanosensors and Modulators of the TME .................................................. 23
  • 1.5.2.2 TRP Channels in Hypoxia and Oxidative Stress ............................................................................ 24
  • PROJECT AIM
  • 2.1 Hypothesis, Objective and Strategy .............................................................................. 29
  • MATERIALS AND METHODS
  • 3.1 Materials ........................................................................................................................ 32
  • 3.1.1 Media ...................................................................................................................................32
  • 3.1.2 Chemicals and Kits ................................................................................................................32
  • 3.1.3 Instruments ..........................................................................................................................34
  • 3.1.4 Oligonucleotides...................................................................................................................35
  • 3.2 Mouse Strains ................................................................................................................ 36
  • 3.2.1 Pancreatic Stellate Cells .......................................................................................................36
  • 3.3 Cell Biological Methods ................................................................................................. 37
  • 3.3.1 Reactivating Frozen RLT-PSCs ..............................................................................................37
  • 3.3.2 Isolation of Mice Pancreatic Stellate Cells ...........................................................................37
  • 3.3.2.1 Identification of mPSCs ................................................................................................................ 38
  • 3.3.3 Cell culture, Passaging and Cell Count .................................................................................39
  • 3.4 Molecular Biological Methods ...................................................................................... 39
  • 3.4.1 Protein Biochemical Methods ..............................................................................................39
  • 3.4.1.1 Protein Isolation and Estimation .................................................................................................. 39
  • 3.4.1.2 Protein Detection and Analysis .................................................................................................... 40
  • 3.4.2 RNA Extraction and Purification from PSCs ..........................................................................43
  • 3.4.3 Reverse Transcriptase PCR ...................................................................................................43
  • 3.4.4 Polymerase Chain Reaction (PCR) ........................................................................................44
  • 3.4.5 DNA Electrophoresis ............................................................................................................45
  • 3.4.6 Quantitative Real-time PCR ..................................................................................................46
  • 3.5 In Vitro Migration Assays .............................................................................................. 48
  • 3.5.1 Hypoxia treatment ...............................................................................................................48
  • 3.5.2 Two-Dimensional Migration .................................................................................................49
  • 3.5.3 Three-Dimensional Migration ..............................................................................................50
  • 3.5.4 Chemotaxis Assay .................................................................................................................52
  • 3.5.5 Autocrine Stimulation Assay ................................................................................................53
  • 3.5.6 Analysis of Migration Data ...................................................................................................53
  • 3.6 Oxygen Consumption and Extracellular Acidification Measurements ...................... 55
  • 3.6.1 Estimation of Oxygen consumption in 3D chamber.............................................................56
  • 3.7 Manganese Quenching .................................................................................................. 58
  • 3.8 Calcium Entry................................................................................................................. 60
  • 3.9 Calpain Activity .............................................................................................................. 60
  • 3.10 Statistical Analysis ...................................................................................................... 62
  • RESULTS
  • 4.1 mPSC Identification ....................................................................................................... 63
  • 4.2 Quantitative Assessment of TRPC Channel Expression in mPSCs .............................. 64
  • 4.3 mPSC Migration in Three Dimensional Space ............................................................. 65
  • 4.3.1 Oxidative Cell Stress in Three Dimensional mPSC Migration ...............................................66
  • 4.4 Hypoxia and mPSC Migration ....................................................................................... 68
  • 4.4.1 Chemically Induced Hypoxia Stimulates mPSC Migration ...................................................68
  • 4.4.1.1 DMOG Induces mPSC Migrat