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Publié par | christian-albrechts-universitat_zu_kiel |
Publié le | 01 janvier 2011 |
Nombre de lectures | 21 |
Langue | Deutsch |
Poids de l'ouvrage | 6 Mo |
Extrait
Aus dem Institut für Klinische Molekularbiologie
(Direktor: Prof. Dr. Schreiber)
der Christian-Albrechts-Universität zu Kiel
Fundamental differences of the hematopoietic growth factors
Flt3L and GM ‐CSF on experimental colitisd epend on
type I‐IFN mediated antimicrobial effects on
intestinal immune response.
Inauguraldissertation
zur
Erlangung der Doktorwürde
der Medizinischen Fakultät
der Christian-Albrechts-Universität zu Kiel
vorgelegt von
Konrad Arnold Altfrid Aden
aus Essen
Kiel 2011
1. Berichterstatter: Prof. Dr. Philip Rosenstiel
2. : Prof. Dr. Jobst Sievers
Tag der mündlichen Prüfung: 22.3.2011
Zum Druck genehmigt, Kiel, den 22.3.2011
Gez.: Cascorbi
Ich versichere hiermit an Eides Statt, dass meine Dissertation mit dem Thema:
Fundamental differences of the hematopoietic growth factors Fms‐like
tyrosine 3‐kinase ligand and GM ‐CSF on experimental colitisd epend
on type I‐IFN mediated antimicrobial effects on intestinal immune
response.
abgesehen von Ratschlägen meines Doktorvaters und meiner sonstigen akademischen Lehrer,
nach Form und Inhalt meine eigene Arbeit ist, dass ich außer den in der Arbeit aufgeführten
keine weiteren Hilfsmittel benutzt habe. Teile dieser Ergebnisse wurden als Poster auf der
Digestive Disease Weak (DDW) 2008, San Diego sowie auf der DDW 2010, New Orleans
präsentiert.
Ich widerspreche nicht nach §5, Abs. 2k der Promotionsordnung der Teilnahme von Zuhörern
an der mündlichen Doktorprüfung.
Kiel, den 4.3.2010
Meinen Eltern
Introduction I
Content
Content ................................................................................. I
Abbreviations ................................. III
Figure Legend ... V
Table Legend ........................................ V
I. Introduction ... 1
Crohn’s Disease and Ulcerative Colitis ............................................................... 1
Introduction: ..................... Fehler! Textmarke nicht definiert.
Clinical and pathologic features ......................... 1
Epidemiology ................................ .............................. 2
Environmental factors ................................ ................................ ............ 3
Genetics ................................ ......... 3
Intestinal immunity and pathophysiology of Crohn’s Disease ................... 6
Mucosal Barrier ................................ ................................ ......................... 6
Innate Immunity in Crohn’s disease ................................. ................ 7
The implications of hematopoietic growth factors on intestinal inflammation. ................. 9
GM ‐CSF as a therapeutic agent in IBD. ............. 9
The role of dendritic cells in the intestinal immune response ................................ ........................... 10
Dendritic cell amplification as a therapeutic target in intestinal inflammation ......................... 11
Fms‐like tyrosine kinase 3 Ligand (Flt3L) ................................ ................................ .. 11
Experimental outline ............................................................. 12
II. Materials and methods .......................................... 14
Animals ....................... 14
Induction and evaluation of experimental colitis using Dextran Sodium Sulfate (DS 14S)
Clinical scoring of DSS colitis ................................ ............................ 14
Macroscopic and histological assess ment of colonic inflammation ................. 15
Myeloperoxidase Assay ................................ ................................ ....... 15
Celllculture ................................................ 16
General conditions for working with cellcultures ................................ ................... 16
Celllines ...... 17
Primary cells ................................ ................................ ............................ 17
Isolation of splenocytes from mice spleen ................................ ................................ .. 17
+Isolation of CD11c Dendritic Cells using magnetic cell sorting (MACS ®) ... 18
Gentamicin protection assay ............................. 19
Working with Nucleid Acids ................................................................................ 20
Total RNA Isolation from cell culture and whole tissues ...... 20
+Isolation of poly A messenger RNA using the Oligotex® mRNA Kit .............. 21
Reverse Transcription ......... 22
Polymerase chain reaction ................................ . 23
Whole genome expression profiling using Agilent chips ................................ ...... 25
Immunohistochemical Methods ........ 27
Flow Cytometry ................................ ................................ ...................... 27
Immunohistochemistry ................................ ....... 28
Statistical Analysis ................................ ................. 29
III. Results ....................................................................... 30
Characterisation of GM ‐CSF and Flt3L effects on DC growth in mice in vivo ...................... 30
Immunophenotype of splenic DC .................... 30
Growth effects on intestinal DC ....................... 32
Evaluation of Flt3L effects in mouse models of acute colitis 33
DSS‐induced acute colitis in balb/c mice. ................................ .... 33
II Introduction
‐/‐DSS‐induced acute colitis in RAG1 mice ................................ ................................ ................................ ... 38
Functional analysis of GM ‐CSF and Flt3L treatment in mice in vivo . ..... 41
Priming effects of GM ‐CSF and Flt3L on TLR ‐9 stimulation 41
Priming effects of GM ‐CSF and Flt3L on TLR ‐7 stimulation 44
Characterization of IFN‐I effects on intestinal immunity .......................................................... 47
Whole mouse genome transcription profiling 47
Real Time PCR confirmation of IFN‐I dependent gene induction ..................... 50
Evaluation of the gentamicin protection assay ......................... 52
GM ‐CSF, but not Flt3L, enhances antimicrobial peptide gene induction in a TLR ‐9 and