A new role of transcription factor SOX17 as potential interaction partner of KLF4 and EGR-1 in human coronary artery smooth muscle cells and in differentiating mouse ES-cells [Elektronische Ressource] / vorgelegt von Nicola Liefold

Nicola Liefold

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 42
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

A new role of transcription factor SOX17
as potential interaction partner
of KLF4 and EGR-1
in human coronary artery smooth muscle cells
and in differentiating mouse ES-cells









Dissertation
der Fakultät für Biologie
der Ludwig-Maximilian-Universität München







Vorgelegt von
Nicola Liefold
aus Osnabrück

am 14.03.07






angefertigt
am GSF-Forschungszentrum
für Umwelt und Gesundheit




Die Arbeit wurde am Institut der Klinischen Molekularbiologie des
Hämatologikums der GSF München angefertigt.










































1. Gutachter: Prof. Dr. Dirk Eick
2. Gutachter: Dr. Stefan Müller

Tag der mündlichen Prüfung: 14.06.07



Table of contents


1. Introduction ....................................................................................................... 1
1.1. The vascular network ............................................................................................... 1
1.1.1. The structure of blood vessels............................................................................ 1
1.1.2. Vasculogenesis ................................................................................................... 2
1.1.3. Angiogenesis ...................................................................................................... 2
1.2. Angiogenic Factors .................................................................................................. 3
1.2.1. Vascular Endothelial Growth Factor (VEGF) and its biological functions ....... 3
1.2.2. The Fibroblast Growth Factors (FGFs).............................................................. 5
1.2.3. The angiopoietins and their receptor Tie2.......................................................... 5
1.2.4. The Hepatocyte Growth Factor (HGF) .............................................................. 6
1.2.5. The Platelet Derived Growth Factor (PDGF) .................................................... 6
1.2.6. The ephrin ligands and their receptors ............................................................... 7
1.3. Vascular processes in embryonic development ....................................................... 8
1.3.1. Vascular development along the mammalian body axis.................................... 8
1.3.2. Vascular processes in the developing liver and prancreas ................................. 8
1.3.3. Vascular processes in the developing kidney..................................................... 9
1.3.4. Vascular processes in placental development .................................................... 9
1.4. Pathological Angiogenesis ....................................................................................... 9
1.5. Vascular cells ......................................................................................................... 11
1.5.1. Endothelial progenitor cells.............................................................................. 11
1.5.2. Mature endothelial cells ................................................................................... 12
1.5.3. Vascular Smooth Muscle Cells (VSMCs)........................................................ 13
1.6. The Sox (Sry box) proteins .................................................................................... 16
1.6.1. Sox protein subgroup F – Sox 7, Sox 17 and Sox 18....................................... 18
1.7. Krüppel-like Factor 4 (KLF4) ................................................................................ 21
1.8. Early Growth Response Factor 1 (EGR-1)............................................................. 23
1.9. The FunGenES project ........................................................................................... 24
1.9.1. ES-cell differentiation mimics embryonic development.................................. 25
1.9.2. Endoderm, Mesoderm and Ectoderm arise from the inner mass of the
blastocyst .......................................................................................................... 26
1.10. Goal of the thesis.................................................................................................... 28
2. Materials........................................................................................................... 29
2.1. Plasmids ................................................................................................................. 29
2.2. Cloning primers...................................................................................................... 29
2.3. Bacteria and Cell lines............................................................................................ 29
2.4. Cell culture media .................................................................................................. 30
2.5. Antibodies .............................................................................................................. 30
2.6. Chemicals and Enzymes......................................................................................... 31
2.7. Working materials .................................................................................................. 31
3. Methods............................................................................................................ 33
3.1. Cell culture ............................................................................................................. 33
3.1.1. Cell passaging .................................................................................................. 33
3.1.2. Differentiation of CGR8 ES-cells .................................................................... 33
3.1.3. Transient Cell Transfection.............................................................................. 34
3.2. Molecular biology techniques ................................................................................ 34
3.2.1. Cloning strategy ............................................................................................... 34
3.2.2. Ligation ............................................................................................................ 34
3.2.3. Transformation of DNA in bacteria ................................................................. 35

3.2.4. Mini-preparation of Plasmid DNA................................................................... 35
3.2.5. Maxi-Preparation of Plasmid DNA from bacteria ........................................... 35
3.2.6. RNA-Isolation .................................................................................................. 36
3.2.7. Reverse Transcription (RT) PCR ..................................................................... 36
3.2.8. The Polymerase Chain Reaction (PCR) ........................................................... 37
3.2.9. Agarose gel electrophoresis ............................................................................. 37
3.3. Immunofluorescence .............................................................................................. 37
3.4. Cell stimulation assays ........................................................................................... 38
3.5. Luciferase-Assays .................................................................................................. 38
3.6. SDS-gel protein electrophoresis............................................................................. 39
3.7. Protein immunoprecipitation.................................................................................. 40
3.8. Co-Immunoprecipitations....................................................................................... 40
4. Results ............................................................................................................. 41
4.1. A new role for SOX17 as potential interaction partner of EGR-1 and KLF4 in
human coronary artery smooth muscle cells .......................................................... 41
4.1.1. Expression of Sox7, Sox17 and Sox18 in different mouse and human tissues in
vivo.................................................................................................................... 41
4.1.2. Expression profile of Sox7, 17 and 18 in different vascular cells in vitro....... 43
4.1.3. Expression of Subgroup F Sox proteins in different mouse tissues in vivo ..... 44
4.1.4. Response of Subgroup F Sox proteins to different stimuli in vascular cells.... 47
4.1.5. SOX17 is upregulated in proliferative conditions in human coronary artery
smooth muscle cells.......................................................................................... 52
4.1.6. EGR-1 induces SOX17 expression in human coronary artery smooth muscle
cells.........................................................