Analysis of the Temporal-Spatial Activation of Wnt-Signaling within Type 1/ Type 2 Immunity during Wound Healing

Die Wundheilung im Zusammenhang mit der Aktivierung des Wnt-Signalweg und Type 1 / Type 2 Immunity wurde im Maus Modell und in-vitro an verschiedenen Zeitpunkten untersucht. Es hat sich herausgestellt, dass der Wnt-Signalweg während der Spätphase der Wundheilung aktiviert wird, also unter Type 2 Immunity. Diese Arbeit deutet darauf hin, dass die Wundheilung über eine Modellierung des Wnt-Signalweg beschleunigt oder verbessert werden kann, allerdings nur an spezifischen Zeitpunkten.:LIST OF ABBREVIATIONS........................................................................................ 5 INDEX OF FIGURES..................................................................................................7 INDEX OF TABLES.....................................................................................................8 SUMMARY.................................................................................................................9 1 INTRODUCTION..............................................................................................11 1.1 Cutaneous wound healing...............................................................................11 1.2 Phases of wound healing................................................................................11 1.2.01 Inflammation............................................................................................11 1.2.02 Proliferation..............................................................................................13 1.2.03 Remodeling..............................................................................................16 1.3 Wnt-signaling...................................................................................................17 1.3.01 Wnt-ligands biogenesis and receptors....................................................17 1.3.02 Canonical and non canonical Wnt signaling...........................................20 1.3.03 Canonical Wnt/ß catenin signaling..........................................................20 1.4 Wound healing and canonical Wnt/ß catenin signaling...................................21 2 AIMS OF THE STUDY......................................................................................25 3 MATERIALS AND EXPERIMENTAL PROCEDURES........................................27 3.1 MATERIALS.....................................................................................................27 3.1.01 Instruments..............................................................................................27 3.1.02 Software..................................................................................................27 3.1.03 Websites..................................................................................................28 3.1.04 Consumables...........................................................................................28 3.1.05 Media and Buffers...................................................................................29 3.1.06 Reagents..................................................................................................29 3.1.07 Stimulation reagents................................................................................30 3.1.08 Anesthesia and analgesia used for the full-thickness skin wound model.......................................................................................................................30 3.1.09 Kits...........................................................................................................30 3.1.10 Quantitative reverse-transcription PCR primers......................................31 3.2 EXPERIMENTAL PROCEDURES.....................................................................32 3.2.01 Mice.........................................................................................................32 3.2.02 Cell Lines.................................................................................................32 3.2.03 Cell counting with trypan blue stain and hemocytometer method.........32 3.2.04 Preparation of murine H133WT immortalized primary keratinocytes cultures....................................................................................................................33 3.2.05 Preparation of RAW264.7 murine macrophages cultures.......................33 3.2.06 Preparation of primary murine fibroblasts cultures.................................33 3.2.07 Type 1 and type 2 immunity cell stimulation...........................................34 3.2.08 Cell RNA isolation....................................................................................34 3.2.09 Mice wounding........................................................................................35 3.2.10 Whole skin tissue isolation......................................................................36 3.2.11 Epidermal and dermal tissue isolation....................................................36 3.2.12 Tissue RNA isolation...............................................................................36 3.2.13 cDNA synthesis.......................................................................................36 3.2.14 Quantitative Real-Time PCR (qPCR).......................................................37 3.2.15 Statistical analysis...................................................................................37 4 EXPERIMENTAL RESULTS..............................................................................39 4.1 Canonical Wnt/ß-catenin signaling activity in different skin cell populations under type 1 and type 2 immunity cytokines stimulation in vitro............................39 4.2 Canonical Wnt/ß-catenin signaling activity under type 1 and type 2 immunity conditions during wound healing in vivo.................................................................42 4.3 Wnt-ligands expression in different skin cell populations under type 1 and type 2 immunity cytokines stimulation in vitro.........................................................44 4.4 Wnt ligands expression in epidermal and dermal fraction under type 1 and type 2 immunity conditions in vivo..........................................................................51 5 DISCUSSION...................................................................................................53 6 CONCLUSIONS...............................................................................................59 7 PERSPECTIVES...............................................................................................61 8 REFERENCES..................................................................................................63 9 APPENDIX.......................................................................................................71 9.1 Supplementary results.....................................................................................71 9.2 Erklärung über die eigenständige Abfassung der Arbeit.................................73 9.3 Curriculum Vitae..............................................................................................75 9.4 Acknowledgments...........................................................................................7