Anti-inflammatory mechanisms of the neutrophil- released antimicrobial peptide α-defensins
نویسنده
چکیده
...................................................................................................................................... I DECLARATION ................................................................................................................................ III ACKNOWLEDGEMENTS ............................................................................................................... IV CONTENTS .................................................................................................................................... VI ABBREVIATIONS .............................................................................................................................. X LIST OF TABLES ........................................................................................................................... XII LIST OF FIGURES ........................................................................................................................ XIII CHAPTER 1: INTRODUCTION ....................................................................................................... 1 1.1 Inflammation during injury ................................................................................................. 2 1.2 Inflammation regulation .................................................................................................... 11 1.3 Key concepts ..................................................................................................................... 15 1.3.1 Autophagy ............................................................................................................... 15 1.3.2 Macrophage activation through R848 stimulation ................................................... 17 1.3.3 Rheumatoid Arthritis ............................................................................................... 19 1.4 Aims of this thesis ............................................................................................................. 20 CHAPTER 2: MATERIALS AND METHODS .............................................................................. 22 2.1 Reagents ............................................................................................................................ 22 2.2 Cell culture ........................................................................................................................ 22 2.2.1 Human Monocyte Derived Macrophage (HMDM) derivation ................................ 22 2.2.2 Generation of human necrotic neutrophils (NN) ..................................................... 23 2.2.3 Bone Marrow Derived Macrophages (BMDMs) ..................................................... 23 2.2.4 Cell lines .................................................................................................................. 24 2.2.5 α-defensin peptides .................................................................................................. 24 2.2.6 HMDM and THP-1 stimulants ................................................................................ 25 2.2.7 HNP1 treated POPC liposomes ............................................................................... 27 2.3 Colourimetric assays ......................................................................................................... 27 2.3.1 Enzyme Linked Immunosorbant Assay (ELISA) .................................................... 27 2.3.2 Alamar Blue assay ................................................................................................... 28 2.4 Protein purification and determination .............................................................................. 29 2.4.1 Bio-Rad protein assay .............................................................................................. 29 2.4.2 Trichloroacetic acid (TCA) protein precipitation .................................................... 30 2.5 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting ............................................................................................................................. 30
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