Impact of bile acids on development of Barrett's esophagus and its progression to esophageal adenocarcinoma [Elektronische Ressource] : the role of apoptosis, COX-2, {NF-_k63B [NF-Kappa-B], reactive oxygen species and CDX-2 / vorgelegt von Grzegorz Burnat

Grzegorz

Sujets

• Gesundheit

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 30
• Poids de l'ouvrage: 2 Mo

Extrait

Impact of bile acids on development of Barrett’s esophagus and its
progression to esophageal adenocarcinoma: the role of apoptosis, COX-2,
NFκB, reactive oxygen species and CDX-2.








Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-
Nürnberg zur
Erlangung des Doktorgrades






vorgelegt von

Grzegorz Burnat

aus

Krakau (Polen)










1





Als Dissertation genehmigt
von den Naturwissenschaftlichen Fakultäten
der Universität Erlangen-Nürnberg






















Tag der mündlichen Prüfung: 30.07.2008


Vorsitzender der
der Promotionskommission: Prof.Dr. Eberhard Bänsch
Erstberichterstatter: PD Dr. Robert Slany
Zweitberichterstatter: Prof. Dr. med. Peter Konturek




2





























Dedicated to
my parents









3 Contents


1. Introduction .................................................................................................................8
1.1. Barrett’ esophagus definition and epidemiology ..............................................8
1.2. Barrett’s esophagus - current definition.......................................................9
1.3. Epidemiology ...........................................................................................10
1.4. Risk factors for Barrett’s esophagus..........................................................10
1.4.1. GERD.....................................................................................................11
1.4.1.1. Symptoms of GERD .............................................................................11
1.4.1.2. Pathogenesis of GERD..........................................................................12
1.4.2 Obesity ...................................................................................................13
1.4.3. Smoking .................................................................................................13
1.5. Duodendogastroesophageal reflux-DGER.................................................14
1.5.1. Bile acids- synthesis and circulation........................................................15
1.5.2. Bile salts as potent factors in development of intestinal metaplasia of the
esophagus.............................................................................................................16
1.5.3. Barrett’s metaplasia an intermediate stage in development of the
esophagealcancer………………………………………………………………...18
1.6. Diagnosis of Barrett’s esophagus ..............................................................19
1.6.1. Endoscopy ..............................................................................................19
1.6.2. Histology................................................................................................21
1.6.3. Other methods useful in diagnosis of Barrett’s esophagus .......................23
1.7. Treatment options of Barrett's esophagus ..................................................24
1.7.1. Antacids..................................................................................................24
1.7.2. Histamine receptor antagonists (H RA) ..................................................25 2
1.7.3. Proton pomp inhibitors............................................................................25
1.7.4. Photodynamic therapy (PDT)..................................................................25
1.7.5. Endoscopic mucosal resection (EMR).....................................................26
1.7.6. Thermal ablation (APC)..........................................................................26
1.7.7. Esophagectomy.......................................................................................26
1.8. Molecular events during development of Barrett's esophagus and Barrett'
adenocarcinoma ...................................................................................................27
1.9. Inflammation as an important initiating factor in the Barrett’s esophagus
carcinogenesis ......................................................................................................28
1.9.1. Cytokines..............................................................................................29
1.9.2. Reactive oxygen species (ROS) ..............................................................30
4 Contents
1.9.3. Nuclear factor κ B (NFκB) .....................................................................31
1.9.4. Cyclooxygenase (COX) and prostaglandins ............................................33
1.9.5. Peroxisome proliferator-activated receptor (PPAR) ............................36
1.10. Angiogenesis ..............................................................................................37
1.11. Proliferation and cell cycle..........................................................................38
1.12. Apoptosis ...................................................................................................40
1.12.1. The Extrinsic Pathway ............................................................................42
1.12.2. The Intrinsic Pathway .............................................................................42
1.13. The molecular changes of DNA.................................................................43
1.14. Other factors...............................................................................................44
1.14.1. Ghrelin...................................................................................................44
1.14.2. Adiponectin ...........................................................................................45
1.14.3. Caudal type homebox transcription factor (CDX)...................................46
2. Aims ....................................................................................................................47
3. Materials and methods.......................................................................................48
3.1. Materials...................................................................................................48
3.1.1. Cell culture .............................................................................................48
3.1.2. Chemicals for RNA isolation: .................................................................49
3.1.3. Complementary DNA synthesis: .............................................................49
3.1.4. Polymerase chain reaction.......................................................................49
3.1.5. Chemicals for DNA electrophoresis ........................................................51
3.1.6. Chemicals and reagents for Western Blot................................................52
3.1.7. Immunohistochemical staining................................................................53
3.1.8. Laboratory equipments ...........................................................................54
3.1.9. Software .................................................................................................54
3.2. Methods....................................................................................................55
3.2.1. 24 hour pH and bile monitoring ..............................................................55
3.2.2. Human biopsies collection ......................................................................56
3.2.3. Isolation of total RNA from biopsies and cell culture. .............................56
3.2.4. Quantification of total RNA....................................................................57
3.2.5. Reverse transcription-polymerase chain reaction (RT-PCR), generation of
complementary DNA ...........................................................................................57
3.2.6. Polymerase chain reaction PCR ..............................................................58
3.2.7. Gel electrophoresis .................................................................................59
5 Contents
3.2.8. Real-Time PCR.......................................................................................59
3.2.9. Western blotting .....................................................................................60
3.2.9.1. Protein isolation. ...................................................................................60
3.2.9.2. Preparation nuclear extract of proteins ..................................................61
3.2.9.3. Bicinchoninic Acid (BCA) Protein Assay .............................................62
3.2.9.4. Gels preparation and proteins electrophoresis........................................62
3.2.9.5. Electrophoretic transfe