C-Myc expression in adult and embryonic endothelial cells [Elektronische Ressource] / vorgelegt von Enikö Kókai

Sujets

• Biologie

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2010
• Nombre de lectures: 14
• Poids de l'ouvrage: 5 Mo

Extrait

C-MYC EXPRESSION IN ADULT AND EMBRYONIC
ENDOTHELIAL CELLS





Dissertation
zur Erlangung des Doktorgrades (Dr. rer. nat.)
an der Fakultät für Naturwissenschaften
der Universiät Ulm


vorgelegt von
ENIKÖ KÓKAI
aus
BUDAPEST, UNGARN



2010



Dekan:
Prof. Dr. Axel Groß, Institut für Theoretische Chemie, Universität Ulm


Erstgutachter:
Prof. Dr. Thomas Wirth, Institut für Physiologische Chemie, Universität Ulm


Zweitgutachter:
Prof. Dr. med. Karl Lenhard Rudolph, Institut für Molekulare Medizin,
Universität Ulm



Tag der Promotion: 25.11.2010












Die Arbeiten im Rahmen der vorliegenden Dissertation wurden in der Institut für
Physiologische Chemie der Universität Ulm durchgeführt und von Herrn Prof. Dr.
Thomas Wirth betreut.

CONTENTS

ZUSAMMENFASSUNG .............................................................................................. 1
SUMMARY .............................................................. 3
1. INTRODUCTION ................................................. 4
1.1. Structure and function of the vascular system ................................................. 4
1.2. Development of the vascular system ............................................................... 8
1.2.1 Vasculogenesis ....................................... 8
1.2.2 Angiogenesis ......................................... 10
1.2.3 Pruning and remodeling ........................ 10
1.2.4 Maturation and remodeling .................... 11
1.2.5 Lymphangiogenesis.................................................................................. 11
1.3. Molecular regulation of vascular development ............................................... 13
1.3.1 Establishing the embryonic vasculature ................................................... 13
1.3.2 Molecules regulating blood vessels branching, remodeling, maturation ... 15
1.4. The C-Myc proto-oncogene ........................ 19
1.4.1 Structure, function and transcriptional regulation of c-Myc ....................... 19
1.4.2 C-Myc and cell proliferation ...................................................................... 21
1.4.3 Gene targets of c-Myc ........................... 22
1.5. C-Myc in vascular development .................. 24
THE AIMS OF THE STUDY .................................. 26
2. MATERIALS AND METHODS ........................... 27
2.1. Transgenic mice ............................................................................................. 27
2.2. Animal experiments..................................... 28
2.2.1 Gross embryonic pathology ................... 28
2.2.2 Gross pathology of adult animals ............................................................. 28
2.2.3 Doxycycline treatment of tumor bearing adult mice .................................. 28
2.2.4 Tumor cell line transplantation .................................................................. 28
2.3. Genotyping of transgenic mice .................... 29
2.3.1 Genomic DNA isolation ......................... 29
2.3.2 Polymerase Chain Reaction (PCR) .......................................................... 29
2.4. Detection of gene expression ...................... 30
2.4.1 Reporter gene detection ........................................................................... 30
2.4.2 Analysis of mRNA expression levels ........................................................ 31

2.4.3 Quantitative real-time PCR analysis ......................................................... 32
2.5. Protein analysis .............................................................................................. 32
2.5.1 Protein concentration measurement by Bradford method ........................ 32
2.5.2 Western immunoblot .............................. 33
2.5.3 VEGF-A ELISA ...................................... 33
2.5.4 Total MMP-9 ELISA .................................................................................. 33
2.6. Flow cytometry analysis .............................. 34
2.6.1 Isolation of primary embryonic endothelial cells ....................................... 34
2.6.2 Detection of apoptosis and proliferation by flow cytometry ....................... 34
2.6.3 Fluorescence Activated Cell Sorting (FACS) ............................................ 35
2.7. Histological analyses...................................................................................... 35
2.7.1 Immunohistochemistry on tissue sections ................................................ 35
2.7.2 Immunohistochemistry on tissue peaces: Whole-mount analyses ........... 37
2.7.3 Electron Microscopy .............................. 39
2.8. Quantitative analysis of immunohistological stainings.................................... 39
2.8.1 Staining quantification .............................................................................. 39
2.8.2 Quantitative analysis of dermal vascular architecture............................... 40
2.9. Materials ..................................................... 42
2.9.1 General chemicals ................................. 42
2.9.2 General buffers and solutions ................ 42
2.9.3 Histology chemicals and materials ........................................................... 47
2.9.4 Special consumption items and equipment .............................................. 48
2.9.5 Antibodies ................................................................................................. 50
2.9.6 The list of primers used for RT-PCRs ....................................................... 51
3. RESULTS .......................................................... 53
3.1. Model for conditional c-Myc expression in endothelial cells ........................... 53
3.2. Endothelial cell-specific c-Myc expression in adult mice ................................ 55
3.2.1 Characterisation of the Tie2-tTA/tetO-Myc adult animals ......................... 55
3.2.2 Transgene expression in Tie2-tTA/tetO-Myc adult animals ...................... 55
3.2.3 Survival of Tie2-tTA/tetO-Myc adult animals ............................................ 55
3.2.4 Gross pathology of Tie2-tTA/tetO-Myc animals ........................................ 57
3.2.5 Tumor classification and statistics ............................................................ 58
3.2.6 Tumor regression upon transgene inactivation ......................................... 62
3.2.7 Establishment of tumor cell lines ........... 63

3.2.8 Characterization of the tumor cell line ...................................................... 63
3.2.9 Subsummary I .......................................................................................... 67
3.3. Characterisation of Tie2-tTA/tetO-Myc embryos ............................................ 68
3.3.1 Transgene expression in Tie2-tTA/tetO-Myc embryos ............................. 68
3.3.2 Endothelial cell-specific c-Myc expression causes embryonic lethality..... 70
3.3.3 Possibly origins of vascular permeability .................................................. 73
3.3.4 Analysis of the lymphatic vessels ............................................................. 77
3.3.5 Analyses of dermal blood vessel architecture .......................................... 79
3.3.6 Electron microscopical analysis of embryonic endothelium ...................... 83
3.3.7 Quantification of endothelial cell apoptosis and proliferation .................... 85
3.3.8 Isolation of embryonic endothelial cells .................................................... 87
3.3.9 Angiogenic modulators expression in purified embryonic endothelial cells . 90
3.3.10 Subsummary II ....................................................................................... 93
4. DISCUSSION .................................................... 94
4.1. C-Myc expression in endothelial cells of adult mice ....................................... 94
4.1.1 C-Myc expression in Tie2-manner in adult mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4 
4.1.2 C-Myc inactivation in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 94
4.1.3 Mouse model for human diseases ............................................................ 95
4.1.4 Animal model for Kaposi's sarcoma ...........96
4.2. C-Myc expression in embryonic endothelial cells ........................................... 97
4.2.1 Possible origins of vascular permeability .................................................. 97
4.2.2 Vascular permeability protecting factors ................................................. 101
4.2.3 Factors modulating vessel remodeling ................................................... 102
4.2.4 C-Myc and angiogenic switch ................................................................. 104
4.2.5 C-Myc: apoptosis and/or proliferation? ......104
4.2.6 The role of c-Myc in embryonic vascular development ........................... 106
4.