Influence of atrial natriuretic peptide on inflammatory pathways in the lung [Elektronische Ressource] / Elke Koch

Elke Koch

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München










Influence of Atrial Natriuretic Peptide on
inflammatory pathways in the lung





Elke Koch

aus Nagold

2006

Erklärung:
Diese Dissertation wurde im Sinne von § 13 Abs.3 bzw. 4 der Promotionsordnung
vom 29.Januar 1998 von Frau Prof. Dr. Angelika M. Vollmar betreut.


Ehrenwörtliche Versicherung:
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.


München, am 16.2.2006










Dissertation eingereicht am: 16.2.2006
1. Gutachter: Prof. Dr. Angelika M. Vollmar
2. Gutachter: PD Dr. Carsten Culmsee
Mündliche Prüfung am: 24.3.2006

























dedicated to my family
Contents
1 Contents

1 Contents .......................................................................................................................... 1
2 Introduction ..................................................................................................................... 7
2.1 Background and aim of the work........................................................................ 8
2.2 Atrial natriuretic peptide (ANP) ......................................................................... 10
2.2.1 Discovery of natriuretic peptide family .................................................... 10
2.2.2 Structure and synthesis of ANP .............................................................. 10
2.2.3 Receptors and signal transduction.......................................................... 11
2.2.4 Effects of ANP on blood pressure ........................................................... 13
2.2.5 Effect of ANP on the immune system ..................................................... 13
2.2.6 Effects of ANP on the lung ...................................................................... 14
2.3 Lung and inflammation ...................................................................................... 16
2.3.1 Overview.................................................................................................. 16
2.3.2 Acute respiratory distress syndrome (ARDS) ......................................... 17
2.3.3 Sepsis...................................................................................................... 18
2.4 Tumour necrosis factor-aaaa (TNF-aaaa) .................................................................... 21
2.4.1 Overview.................................................................................................. 21
2.4.2 Receptors and signalling......................................................................... 21
2.5 Lipopolysaccharide (LPS).................................................................................. 23
2.5.1 Overview.................................................................................................. 23
2.5.2 Receptor and signalling........................................................................... 23
2.6 p38 mitogen activated protein kinase (p38 MAPK) ......................................... 25
2.7 Proteine kinase B / Akt....................................................................................... 26
2.8 Adhesion Molecules ........................................................................................... 27
2.8.1 Overview.................................................................................................. 27
2.8.2 ICAM-1 .................................................................................................... 27
1 Contents
2.8.3 Role of ICAM-1 in lung inflammation....................................................... 28
3 Materials and methods................................................................................................. 29
3.1 Cell culture .......................................................................................................... 30
3.1.1 Materials.................................................................................................. 30
3.1.2 Solutions.................................................................................................. 30
3.1.3 Type II alveolar epithelial cell line A549.................................................. 30
3.1.4 Culture of A549 ....................................................................................... 31
3.1.5 Passaging................................................................................................ 31
3.1.6 Freezing and thawing.............................................................................. 31
3.2 LPS model of murine sepsis.............................................................................. 32
3.2.1 Animals.................................................................................................... 32
3.2.2 Materials and solutions............................................................................ 33
3.2.3 Experimental setting and tissue sample generation ............................... 33
3.2.3.1 TNF-a measurement in plasma and tissue samples....................... 33
3.2.3.2 Experimental setting for tissue sample generation.......................... 34
3.3 Western Blot analysis of protein....................................................................... 35
3.3.1 Sample preparation ................................................................................. 35
3.3.1.1 Solutions .......................................................................................... 35
3.3.1.2 Preparation of whole cell lysates..................................................... 36
3.3.1.3 Preparation of whole organ lysates ................................................. 36
3.3.1.4 Protein determination ...................................................................... 36
3.3.2 Sodium dodecyl sulfate - polyacrylamide gel electrophoresis ................ 37
3.3.2.1 Solutions .......................................................................................... 37
3.3.2.2 Electrophoresis................................................................................ 37
3.3.3 Western Blot............................................................................................ 38
3.3.3.1 Solutions .......................................................................................... 38
3.3.3.2 Antibodies........................................................................................ 39
3.3.3.3 Semi-Dry blotting ............................................................................. 39
2 Contents
3.3.3.4 Protein detection.............................................................................. 40
3.3.3.5 Coomassie blue staining ................................................................. 41
3.3.3.6 Stripping and reprobing ................................................................... 41
3.4 Electro Mobility Shift Assay (EMSA) ................................................................ 41
3.4.1 Solutions.................................................................................................. 41
3.4.2 Isolation of nuclear protein ...................................................................... 42
3.4.2.1 Preparation from cells...................................................................... 42
3.4.2.2 Preparation from lung tissue............................................................ 43
3.4.3 Protein determination .............................................................................. 43
3.4.4 Radioactive labeling of consensus oligonucleotides............................... 44
3.4.5 Binding reaction and electrophoretic separation..................................... 44
3.5 In vitro phosphorylation by p38 MAPK ............................................................ 45
3.5.1 Solutions.................................................................................................. 45
3.5.2 Immunoprecipitation ................................................................................ 46
3.5.3 In vitro phosphorylation assay................................................................. 46
3.6 Isolation and characterization of RNA.............................................................. 47
3.7 Reverse transcription - polymerase chain reaction........................................ 48
3.7.1 Solutions.................................................................................................. 48
3.7.2 Primers .................................................................................................... 48
3.7.3 Reverse transcription and polymerase chain reaction ............................ 49
3.7.4 Agarose gel electrophoresis.................................................................... 49
3.8 Real time PCR ..................................................................................................... 50
3.8.1 Primer and probe.....................................................