Signal transduction pathways induced by the anti psoriatic drug anthralin in cultured human keratinocytes [Elektronische Ressource] / vorgelegt von Astrid Beyerle

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• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2002
• Nombre de lectures: 27
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Aus der Klinik für Dermatologie
der Heinrich-Heine-Universität Düsseldorf
Direktor: Prof. Dr. med. Dr. h.c. Thomas Ruzicka












Signal Transduction Pathways Induced by the Anti-Psoriatic Drug
Anthralin in Cultured Human Keratinocytes





Dissertation
zur Erlangung des Grades eines Doktors der Medizin
Der Medizinischen Fakultät der Heinrich-Heine-Universität Düsseldorf

vorgelegt von

Astrid Beyerle

2002













Als Inauguraldissertation gedruckt mit Genehmigung der
Medizinischen Fakultät der
Heinrich-Heine-Universität Düsseldorf



Gez.: Prof. Dr. med. Dr. phil. Alfons Labisch M.A.
Dekan

Referent: Prof. Dr. med. Dr. h.c. Thomas Ruzicka
Korreferent: Prof. Dr. med. Dr. rer. Bernd Nürnberg




TABLE OF CONTENTS
1 INTRODUCTION............................................................................................5
1.1 PSORIASIS........................................................................................................5
1.1.1 DEFINITION....................................................................................................5
1.1.2 EPIDEMIOLOGY OF PSORIASIS ........................................................................5
1.1.3 ETIOLOGY, PATHOGENESIS AND HISTOPATHOLOGY OF PSORIASIS....................5
1.2 THERAPEUTIC APPROACHES IN THE MANAGEMENT OF PSORIASIS.......................7
1.2.1 SYSTEMIC TREATMENT...................................................................................8
1.2.2 TREATMENT WITH ULTRAVIOLET LIGHT...........................................................9
1.2.2.1 PHOTOTHERAPY .....................................................................................9
1.2.2.2 PHOTOCHEMOTHERAPY (PUVA) .............................................................9
1.2.3 TOPICAL TREATMENTS9
1.3 ANTHRALIN ....................................................................................................10
1.3.1 HISTORICAL REMARKS .................................................................................10
1.3.2 CURRENT USE OF ANTHRALIN......................................................................10
1.3.3 CHEMICAL STRUCTURE AND GENERATION OF REACTIVE OXYGEN SPECIES BY
ANTHRALIN ..................................................................................................10
1.3.4 MECHANISM OF ACTION OF ANTHRALIN .........................................................15
1.4 GENERAL CELLULAR RESPONSE MECHANISMS TO ENVIRONMENTAL STRESSES.16
1.4.1 LIPID PEROXIDATION AND GENERATION OF REACTIVE OXYGEN SPECIES (ROS) ..
...............................................................................................................17
1.4.2 THE EPIDERMAL GROWTH FACTOR (EGF) AND ITS RECEPTOR .......................18
1.4.3 MAP KINASES (MAPKS) ..............................................................................19
1.4.4 C-JUN N-TERMINAL KINASE (JNK)................................................................20
1.5 AIMS OF THE PRESENT STUDY .........................................................................22
2 MATERIALS AND METHODS.....................................................................23
2.1 GENERAL REMARKS .......................................................................................23
2.1.1 MATERIALS..................................................................................................23
2.2 CELL CULTURE...............................................................................................24
2.2.1 CULTIVATION OF HUMAN KERATINOCYTES.....................................................24
2.2.1.1 Cell culture Media...............................................................................24
2.2.1.2 Protocol
2.2.2 ISOLATION AND CULTIVATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS
(PBMC) ......................................................................................................25
2.2.2.1 Cell culture Media25
2.2.2.2 Protocol...............................................................................................
2.2.3 CULTIVATION OF HACAT-CELLS ...................................................................26
2.2.3.1 Cell culture Media26
2.2.3.2 Protocol
2.3 DETECTION OF LIPID PEROXIDATION ...............................................................27
2.3.1 CELLS .........................................................................................................27
2.3.2 REAGENTS AND MATERIALS .........................................................................27
2.3.3 PROTOCOL..................................................................................................27
2.4 LASER SCANNING CONFOCAL MICROSCOPY.....................................................29
2.4.1 CELLS29
2.4.2 REAGENTS AND MATERIALS29
2.4.3 PRINCIPLE OF MODE OF ACTION....................................................................29
2.4.4 PROTOCOL29
2.5 ASSAY OF EXTRACELLULAR HYDROGEN PEROXIDE (H O ) PRODUCTION ..........30 2 2
2.5.1 CELLS .........................................................................................................30
1TABLE OF CONTENTS
2.5.2 REAGENTS AND MATERIALS .........................................................................30
2.5.3 PROTOCOL..................................................................................................30
2.6 CELL DEATH/CELL VIABILITY ASSAYS..............................................................31
2.6.1 PROPIDIUM IODIDE AND DAPI STAINING........................................................31
2.6.1.1 Cells....................................................................................................31
2.6.1.2 Reagents and Materials ......................................................................
2.6.1.3 Protocol...............................................................................................32
2.6.2 LIVE/DEAD® VIABILITY/CYTOTOXICITY ASSAY ............................................
2.6.2.1 Cells32
2.6.2.2
2.6.2.3 Background.........................................................................................33
2.6.2.4 Protocol
2.6.3 CELL DEATH ASSAY FOR PBMC USING TRYPAN BLUE ...................................34
2.6.3.1 Cells....................................................................................................34
2.6.3.2 Reagents and Materials ......................................................................
2.6.3.3 Background34
2.6.3.4 Protocol...............................................................................................
2.7 JNK KINASE ASSAY........................................................................................35
2.7.1 CELLS .........................................................................................................35
2.7.2 REAGENTS AND MATERIALS .........................................................................35
2.7.3 BUFFERS.....................................................................................................35
2.7.3.1 WCE-BUFFER.......................................................................................35
2.7.3.2 B-BUFFER.............................................................................................35
2.7.3.3 HEPES BALANCED BUFFER (HBB) ........................................................36
2.7.3.4 KB-BUFFER ..........................................................................................36
2.7.3.5 SAMPLE BUFFER (2X) ............................................................................36
2.7.4 PROTOCOL..................................................................................................37
2.8 IMMUNOPRECIPITATION OF THE EGF RECEPTOR..............................................39
2.8.1 CELLS .........................................................................................................39
2.8.2 REAGENTS AND MATERIALS .........................................................................39
2.8.3 FRACKELTON BUFFER..................................................................................39
2.8.4 ANTIBODIES.................................................................................................39
2.8.5 PROTOCOL40
2.9 DETECTION OF ACTIVATED ERK1 AND ERK2..................................................41
2.9.1 CELLS41
2.9.2 REAGENTS AND MATERIALS41
2.9.3 BUFFERS.....................................................................................................41
2.9.3.1 TBS (TRIS BUFFERED SALINE PH 7.4)...................................................41
2.9.3.2 SDS RUNNING BUFFER.........................................................................41
2.9.3.3 TRANSFER BUFFER...............................................................................41
2.9.3.4 WASH BUFFER......................................................................................42
2.9.3.5 BLOCKING BUFFER................................................................................42
2.9.3.6 LAEMMLI SAMPLE BUFFER (2X) ....................................