The cardiovascular hormone ANP interferes with LPS-induced early inflammatory pathways in vitro and in vivo [Elektronische Ressource] / Kathrin Ladetzki-Baehs

Kathrin Ladetzki-Baehs

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

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


THE CARDIOVASCULAR HORMONE ANP
INTERFERES WITH LPS-INDUCED
EARLY INFLAMMATORY PATHWAYS
IN VITRO AND IN VIVO










Kathrin Ladetzki-Baehs
aus Gardelegen
2006
Erklärung:
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Prof. Dr. Angelika M. Vollmar betreut.


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

München, 27. März 2006

Kathrin Ladetzki-Baehs








Dissertation eingereicht am: 30.März 2006
1. Gutachter: Prof. Dr. Angelika M. Vollmar
2. Gutachter: Prof. Dr. Martin Biel
Mündliche Prüfung am: 28. April 2006 Index I
1 INTRODUCTION .....................................................................................- 1 -
1.1 BACKGROUND AND AIM OF THE STUDY ...............................................................- 2 -
1.2 NATRIURETIC PEPTIDES ..........................................................................................- 4 -
1.2.1 Natriuretic peptide receptors ........................................................................................... - 5 -
1.2.2 Atrial natriuretic peptide structure and function - 6 -
1.2.3 ANP and the immune system - 8 -
1.2.3.1 Influence of ANP on LPS-induced inflammatory processes............................................- 8 -
1.2.3.2 Influence of ANP on TNF- α-induced inflammatory processes ........................................- 9 -
1.2.3.3 Cytoprotective effects of ANP.........................................................................................- 9 -
1.3 SEPSIS, SEVERE SEPSIS AND SEPTIC SHOCK ...................................................- 10 -
1.3.1 The inflammatory cascade ............................................................................................- 11 -
1.3.2 Signaling mechanisms involved in sepsis..................................................................... - 13 -
1.3.2.1 LPS signaling................................................................................................................- 13 -
Toll-like receptors .........................................................................................................- 13 -
Toptor-4 mediated LPS signaling..................................................................- 14 -
The early response – the MyD88-dependent pathway .................................................- 14 -
1.3.2.2 TNF- α signaling ............................................................................................................- 16 -
TNF- α processing- 16 -
Signal transduction by tumor necrosis factor receptor-1...............................................- 16 -
1.3.2.3 NF- κB ...........................................................................................................................- 18 -
1.3.2.4 Protein kinases .............................................................................................................- 20 -
p38 MAPK....................................................................................................................- 20 -
PI3 kinase - Akt pathway ..............................................................................................- 21 -
1.4 SEPSIS & INFLAMMATION - IN VIVO AND EX VIVO............................................- 22 -
1.4.1 The liver......................................................................................................................... - 22 -
Hepatocytes..................................................................................................................- 23 -
Kupffer cells- 24 -
1.4.2 Blood leukocytes - monocytes and neutrophils.............................................................- 24 -
2 MATERIAL AND METHODS ................................................................- 25 -
2.1 MURINE MODEL OF LPS-INDUCED SEPTIC SHOCK............................................- 26 -
2.1.1 Animals.......................................................................................................................... - 26 -
2.1.2 Material and solutions ...................................................................................................- 26 -
2.1.3 Experimental setting...................................................................................................... - 27 - II Index
2.2 LIVER CELL CULTURE ............................................................................................- 28 -
2.2.1 Animals.......................................................................................................................... - 28 -
2.2.2 Material and solutions ...................................................................................................- 28 -
2.2.3 Isolation of primary hepatocytes ...................................................................................- 30 -
2.2.4 Experimental setting...................................................................................................... - 31 -
2.3 BLOOD.......................................................................................................................- 32 -
2.3.1 Material and solutions- 32 -
2.3.2 Sample generation ........................................................................................................ - 32 -
2.3.2.1 Sample generation of human blood ..............................................................................- 32 -
2.3.2.2 Sample generation of mouse blood- 33 -
2.3.3 Experimental setting...................................................................................................... - 33 -
2.4 WESTERN BLOT ANALYSIS....................................................................................- 35 -
2.4.1 Material and solutions ...................................................................................................- 35 -
2.4.2 Antibodies...................................................................................................................... - 36 -
2.4.3 Sample preparation....................................................................................................... - 37 -
2.4.3.1 Preparation of protein extracts from mouse liver tissue ................................................- 37 -
2.4.3.2 Preparation n extracts from isolated hepatocytes ............................................- 37 -
2.4.3.3 Protein concentration determination .............................................................................- 37 -
2.4.4 Electrophoresis.............................................................................................................. - 38 -
2.4.5 Semi-dry blotting ........................................................................................................... - 38 -
2.4.6 Protein detection - 38 -
2.4.6.1 Specific protein determination on the membrane..........................................................- 38 -
2.4.6.2 Unspecific protein staining with Coomassie blue- 39 -
2.5 EMSA – ELECTRO MOBILITY SHIFT ASSAY .........................................................- 39 -
2.5.1 Material and solutions ...................................................................................................- 39 -
2.5.2 Radioactive labeling of consensus oligonucleotides.....................................................- 40 -
2.5.3 Sample preparation....................................................................................................... - 41 -
2.5.3.1 Preparation of nuclear extracts from mouse liver tissue ...............................................- 41 -
2.5.3.2 Preparation of nuclear extracts from isolated hepatocytes ...........................................- 41 -
2.5.3.3 Protein concentration determination .............................................................................- 41 -
2.5.4 Binding reaction and electrophoretic separation...........................................................- 42 -
2.5.4.1 Control experiments......................................................................................................- 42 -
2.5.5 Detection and evaluation............................................................................................... - 42 -

Index III
2.6 HISTOLOGICAL ANALYSIS .....................................................................................- 43 -
2.6.1 Material and solutions ...................................................................................................- 43 -
2.6.2 Antibodies...................................................................................................................... - 43 -
2.6.3 Histological analysis of liver tissue................................................................................- 44 -
2.6.3.1 Immunhistochemistry....................................................................................................- 44 -
2.6.3.2 Tunel staining and infiltration of leukocytes ..................................................................- 44 -
2.7 REAL TIME RT-PCR..................