HGF/c-Met and IL-6/gp130 mediated signalling pathways in a model of acute and chronic cholestatic liver injury in mice [Elektronische Ressource] / Arne Giebeler

Agiebeler

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

“HGF/c-Met and IL-6/gp130 mediated signalling
pathways in a model of acute and chronic
cholestatic liver injury in mice.”



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der RWTH Aachen University vorgelegte Dissertation zur Erlangung des
akademischen Grades eines Doktors der Naturwissenschaften

vorgelegt von

Diplom-Biologe

Arne Giebeler

aus Lahn-Giessen



Berichter: Universitätsprofessor Dr. rer. nat. Marc Spehr

Universitätsprofessor Dr. rer. nat. Martin Zenke

Tag der mündlichen Prüfung: 31.05.2010



Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.











































Table of Content
Summary............................................................................................................. I
1. Introduction .................................................................................................... 1
1.1. Receptor Tyrosine Kinases ......................................................................... 1
1.2.1. Structure of c-Met and HGF ..................................................................... 1
1.2.2. c-Met mediated Signal transduction ......................................................... 4
1.2.3. c-Met signalling in liver disease................................................................ 7
1.2.4. c-Met as a therapeutic target.................................................................... 8
1.3. The IL-6 cytokine family and its signal transducer gp130.......................... 10
1.3.1 The IL-6 Family ................................................................................ 10
1.3.2. IL-6 signalling.................................................................................. 12
1.2.4 IL-6 and gp130 in liver disease and liver regeneration..................... 14
1.3. Liver fibrosis.............................................................................................. 17
1.4. The Acute Phase Response...................................................................... 21
1.5 The Bile duct ligation model ................................................................ 22
1.6. Aims of this study ...................................................................................... 23
2. Material and Methods................................................................................... 24
2.1. Methods................................................................................................. 24
2.1.1. Bile duct ligation.............................................................................. 24
2.1.2. Serum preparation .......................................................................... 24
2.1.3. DNA-isolation from mouse tails....................................................... 24
2.1.3. Generation of hepatocyte specific knockout mice ........................... 25
2.1.4. Genotyping PCR for Alb-cre, c-Met, gp130..................................... 26
2.1.5. Immunostaining for BrDU................................................................ 27
+2.1.6. Immunostaining for CD4 -cells........................................................ 28
+2.1.7. Immunostaining for F4/80 cells...................................................... 28
+2.1.8. Immunostaining for CD11b cells.................................................... 29
+2.1.9. Immunostaining for Ly6G cells 29
2.1.10. Proof of the depletion of the c-Met on mRNA level ....................... 29
2.1.11. RNA-extraction from total liver 30
2.1.12. Gene expression analysis via Real-Time PCR ............................. 31
2.1.13. Western blotting ............................................................................ 32
2.1.14. Terminal deoxynucleotidyl transferase dNTP nick and labelling
(TUNEL) ................. 33
2.1.15. Germ assay from liver................................................................... 33
2.1.16. Germ-Assay from blood ................................................................ 34
2.1.17. Lysogeny broth (LB) -media and agar plates ................................ 34
2.1.18. Protein extraction from total liver................................................... 34
2.1.19. In vitro stimulation of primary murine hepatocytes with murine HGF
.................................................................................................................. 35
2.1.20. Statistical analysis......................................................................... 35
2.2. Chemicals ................................................................................................. 36
2.2.2. Ready to Use Buffers ............................................................................. 38
2.2.3. Antibodies .............................................................................................. 38
2.3. Buffers ................................................................................................... 39
2.3.1. DNA loading buffer.......................................................................... 39
2.3.2. Tail lysis buffer ................................................................................ 39
2.3.3. Tris buffered saline 39
2.3.4. Phosphate buffered saline .............................................................. 40
2.3.5. Whole Cell Protein Extraction Buffer............................................... 40
2.3.6. Whole Cell Prot 40
2.3.7. Blotting transfer buffer..................................................................... 40 Table of Content
2.3.8. RIPA buffer...................................................................................... 41
2.4.1. Glasware ............................................................................................... 44
2.4.2. Plasticware ............................................................................................. 44
3. Results.......................................................................................................... 46
3.1. Involvement of c-Met during hepatic fibrosis development and antiapoptotic
signalling..................................................................................................... 46
3.1.1. Embryonic hepatocyte specific c-Met deletion is lethal........................... 46
3.1.2. Hepatocyte specific deletion of c-Met results in enhanced necrosis and
fibrosis after bile duct ligation .................................................................. 47
3.1.3. Enhanced inflammatory response and immune cell infiltration after bile
Δhepa duct ligation in c-Met mice ................................................................ 49
3.1.5. Microarray analysis identifies a cluster of c-Met-dependent regulated anti-
apoptotic genes ........................................................................................ 52
∆hepa3.1.7. Bile duct ligation results in more liver fibrosis in c-Met mice ............ 56
3.2. c-Met and gp130 mediated signalling during the acute phase response after
BDL............................................................................................................. 59
3.2.1. Decreased survival of mice deficient in gp130 after BDL........................ 59
3.2.2. Changes in serum parameters during the early phase after BDL ........... 60
3.2.3. Histological analysis after BDL ............................................................... 61
3.2.4. Bacterial load after Bile Duct Ligation..................................................... 63
3.2.5. Dissection of the acute phase response................................................. 66
3.2.7. Hepatic infiltration of immune cells after BDL ......................................... 70
3.2.8. Expression of antimicrobial peptides ...................................................... 73
4. Discussion .................................................................................................... 75
5. References ................................................................................................... 84
Acknowledgements .......................................................................................... 96
Curriculum vitae................................................................................................ 97
Abbreviations 98
Zusammenfassung ......................................................................................... 101
Publications 103
Awards............................................................................................................ 105



















Table of Content

Figures:

Figure 1: Structure of the c-Met receptor tyrosine kinase 3
Figure 2: HGF processing from its inactive precours