Alveolar epithelial cell specific gene expression in vivo [Elektronische Ressource] : effect of TGF-β1 stimulation [[Elektronische Ressource]] / by Julia Sevilla Pérez

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2008
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Alveolar epithelial cell-specific gene expression in vivo:
Effect of TGF-β1 stimulation












Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfillment of the requirements
for the degree of Doctor of Medicine
in the Faculty of Medicine (Human Biology)
of the Justus-Liebig-University of Giessen




by
Sevilla Pérez, Julia
from
Madrid, Spain



Giessen, September 2007



From the Institute of Internal Medicine
of the Justus-Liebig-University of Giessen
Director: Prof. Dr. W. Seeger
















Committee Member: Prof. Dr. Seeger

Committee Member: Prof. Dr. Kracht


th
Date of Doctoral Defense: 16 of April 2008




TABLE OF CONTENTS
I TABLE OF CONTENTS
I TABLE OF CONTENTS.............................................................................................I
II LIST OF FIGURES ................................................................................................. III
III LIST OF TABLES .................................................................................................. V
IV LIST OF ABBREVIATIONS ................................................................................VI
SUMMARY ................................................................................................................IX
ZUSAMMENFASSUNG ............................................................................................. X
1 INTRODUCTION............................................................................................... 12
1.1 ACTIVE SIGNALLING IN THE LUNG ................................................... 13
1.1.1 Transforming growth factor-beta (TGF-β) superfamily...................... 13
1.1.1.1 TGF-β ligands.................................................................................. 14
1.1.1.2 TGF-β receptors: classification and structure.................................. 15
1.1.1.3 Intracellular signalling molecules: Smads....................................... 16
1.1.1.4 Regulation of the TGF-β pathway................................................... 17
1.1.1.5 TGF-β target genes .......................................................................... 18
1.1.1.6 TGF-β transgenic mice .................................................................... 19
1.1.2 Additional signalling cascades active in the lung................................ 20
1.1.2.1 Wnt pathway.................................................................................... 20
1.1.2.2 MAPK pathway: JNK, p38 and ERK MAP kinases ....................... 20
1.1.2.3 NF-κB pathway ............................................................................... 21
1.1.2.4 PI3K/AKT pathway......................................................................... 22
1.2 LUNG ANATOMY, PHYSIOLOGY AND FUNCTION .......................... 22
1.3 THE LUNG EPITHELIUM ........................................................................ 23
1.3.1 Cell types and function........................................................................ 23
1.3.2 Cell type-specific markers................................................................... 26
1.3.3 Lung epithelium-related diseases ........................................................ 27
1.4 TGF- AND EPITHELIAL LUNG DISEASES......................................... 28
1.4.1 Expression and role of TGF-β in the lung........................................... 28
1.4.2 TGF-β-related lung diseases ............................................................... 28
1.4.3 Idiopathic Pulmonary Fibrosis (IPF) ................................................... 30
1.5 STUDIES ON LUNG EPITHELIUM......................................................... 32
2 AIM OF THE STUDY ........................................................................................ 35
3 MATERIALS AND METHODS ........................................................................ 36
3.1 MATERIALS .............................................................................................. 36
3.1.1 General chemicals................................................................................ 36
3.1.2 Cell culture reagents ............................................................................ 37
3.1.3 Primers................................................................................................. 38
3.1.4 Antibodies............................................................................................ 38
3.2 METHODS.................................................................................................. 39
3.2.1 Mammalian cell culture....................................................................... 39
3.2.1.1 Cell isolation.................................................................................... 39
3.2.1.2 Cell culture ...................................................................................... 40
3.2.2 Molecular biology techniques ............................................................. 40
3.2.2.1 Epithelial lavage extraction ............................................................. 40
3.2.2.2 RNA isolation from cultured cells................................................... 41
3.2.2.3 RNA isolation from lung homogenates........................................... 41
3.2.2.4 RNA isolation from epithelial lavage samples................................ 41
3.2.2.5 Reverse transcription (RT) reaction ................................................ 41


I

bTABLE OF CONTENTS
3.2.2.6 Polymerase chain reaction (PCR).................................................... 42
3.2.2.6.1 Semiquantitative PCR .............................................................. 42
3.2.2.6.2 Quatitative PCR (qPCR)............................................................ 43
3.2.2.7 DNA agarose gel electrophoresis .................................................... 44
3.2.2.8 Oligo microarray.............................................................................. 44
3.2.3 Immunological methods ...................................................................... 46
3.2.3.1 Immunohistochemistry.................................................................... 46
3.2.3.2 Western blotting .............................................................................. 47
3.2.4 Animal handling .................................................................................. 48
3.2.4.1 Instillation of substances into the murine lung................................ 48
3.2.4.1.1 Orotracheal administration (OT) ............................................... 49
3.2.4.1.2 Intranasal administration (IN) ................................................... 49
3.2.4.1.3 Intratracheal administration (IT) ............................................... 49
3.2.4.1.4 Microspray (MS) ....................................................................... 49
3.2.4.1.5 Nebulisation (NB) ..................................................................... 49
3.2.5 Statistical analysis ............................................................................... 50
4 RESULTS............................................................................................................ 51
4.1 OPTIMISATION OF LOCAL DELIVERY TO THE LUNG .................... 51
4.2 TGF- 1 ADMINISTRATION .................................................................... 54
4.2.1 Orotracheally-instilled TGF-β1 activates the Smad-dependent pathway
54
4.2.2 TGF-β1-induced gene transcription .................................................... 57
4.2.3 Immunohistochemistry on saline and TGF-β1-treated murine lungs.. 60
4.3 EPITHELIAL LAVAGE............................................................................. 61
4.3.1 Epithelial lavage: optimisation of the guanidinium isothiocyanate
concentration........................................................................................ 62
4.3.2 Epithelial lavage: retention time optimisation..................................... 64
4.3.3 Reproducibility of the epithelial lavage technique.............................. 64
4.3.4 Epithelial lavage effect on the lung morphology................................. 66
4.3.5 Steps to perform the epithelial lavage technique................................. 67
4.3.6 Enrichment of the epithelial lavage fractions...................................... 68
4.4 EPITHELIAL LAVAGE VERSUS PRIMARY ALVEOLAR EPITHELIAL
TYPE II (ATII) CELLS........................................................................................... 69
4.5 MARKER GENE EXPRESSION IN EPITHELIAL LAVAGE SAMPLES
AFTER TGF- 1-INSTILLATION ............................................................. 72
5 DISCUSSION...................................................................................................... 75
5.1 OROTRACHEAL INSTILLATION........................................................... 75
5.2 TGF- 1-INDUCED SIGNAL TRANSDUCTION AND GENE
TRANSCRIPTION..................................................................