Functions of pleiotrophin in the regulation of hematopoiesis [Elektronische Ressource] / Monika Kröger

Monika Graetzer-Tortorella

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 16
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Entwicklungsgenetik


Functions of Pleiotrophin in the regulation of hematopoiesis

Monika Kröger


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. S. Scherer
Prüfer der Dissertation: 1. Univ.-Prof. Dr. W. Wurst
2. Priv.-Doz. Dr. R. A. J. Oostendorp
3. Priv.-Doz. Dr. U. Keller


Die Dissertation wurde am 27.04.2010 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München am
11.10.2010 angenommen.


TABLE OF CONTENTS ·
Table of contents
Zusammenfassung .................................................................................................................. 1
Summary .................................................................................................................................. 3
1. Introduction...................................................................................................................... 3
1.1. Hematopoietic stem cells and their regulation ................................................... 3
1.1.1. The hematopoietic system .............................................................................. 3
1.1.2. The hematopoietic hierarchy ......................................................................... 3
1.1.3. The hematopoietic stem cell niche ................................................................ 5
1.1.4. Cell cycle regulation of HSC .......................................................................... 7
1.1.5. Regulation of HSC function by the microenvironment ........................... 10
1.2. Pleiotrophin ............................................................................................................ 13
1.2.1. Functions of Pleiotrophin ............................................................................. 14
1.2.2. Signalling pathways of Pleiotrophin .......................................................... 17
1.2.3. Pleiotrophin and stem cells .......................................................................... 19
1.3. Aim of this thesis ................................................................................................... 20
2. Materials and methods ................................................................................................. 21
2.1. Materials ................................................................................................................. 21
2.1.1. Mice ................................................................................................................. 21
2.1.2. Cell lines.......................................................................................................... 21
2.1.3. Antibodies ...................................................................................................... 21
2.1.4. Primer .............................................................................................................. 23
2.2. Methods .................................................................................................................. 26
In vivo methods .............................................................................................................. 26
2.2.1. Genotyping ..................................................................................................... 26
2.2.2. Preparation of murine tissues ...................................................................... 28
2.2.3. Transplantation assays ................................................................................. 28
2.2.4. 5-FU and BrdU injections ............................................................................. 29
In vitro methods ............................................................................................................. 30
2.2.5. Cell culture ..................................................................................................... 30
TABLE OF CONTENTS TABLE OF CONTENTS ·
2.2.6. Stable Knockdown of Ptn ............................................................................. 30
-2.2.7. Co-cultures of lin with stromal cell lines .................................................. 31
2.2.8. RNA isolation ................................................................................................. 32
2.2.9. Reverse transcription .................................................................................... 33
2.2.10. Real time PCR ................................................................................................ 33
2.2.11. Western blotting ............................................................................................ 35
2.2.12. Colony forming assay ................................................................................... 36
2.2.13. ELISA .............................................................................................................. 37
2.2.14. Flowcytometry staining ................................................................................ 37
2.2.15. Single cell staining assays ............................................................................ 38
2.2.16. Apoptosis assay ............................................................................................. 39
2.2.17. Statistics .......................................................................................................... 40
3. Results ............................................................................................................................. 41
3.1. Confirmation of array results .............................................................................. 41
3.2. Knockdown of Pleiotrophin in supporting stromal cell lines ......................... 43
3.3. Progenitor frequency in co-cultures lacking Ptn .............................................. 44
3.4. Characterization of 129 Ptn KO mice ................................................................. 46
3.5. Characterization of old 129 Ptn KO mice ........................................................... 56
3.6. Characterization of (129xB6)F2 mice .................................................................. 57
3.7. Proliferation in 129 and (129xB6)F2 Ptn KO mice ............................................ 61
3.8. Characterization of rederived 129 Ptn KO mice. .............................................. 64
3.9. Transplantation of Ptn deficient HSCs ............................................................... 68
3.10. Transplantation into a Ptn-deficient microenvironment ................................. 72
3.11. Transplantation of in vitro co-cultures ............................................................... 80
3.12. Analysis of 2 weeks co-cultures .......................................................................... 84
3.13. Analysis of 3 days cultures .................................................................................. 85
3.14. Gene expression analysis of 3 days co-cultures ................................................ 87
3.15. Analysis of protein levels after 3 days co-culture ............................................. 92
3.16. Analysis of apoptosis in cells lacking microenvironmental Ptn ..................... 95
TABLE OF CONTENTS TABLE OF CONTENTS ·
4. Discussion ..................................................................................................................... 100
4.1. Functions of Pleiotrophin in steady-state hematopoiesis .............................. 101
4.2. Phenotype of 129 Ptn KO mice .......................................................................... 103
4.3. Intrinsic functions of Pleiotrophin .................................................................... 103
4.4. Extrinsic functions of Pleiotrophin ................................................................... 104
4.5. Mechanistical insights into the molecular basis of Ptn functions ................ 109
4.6. Functions of Ptn in granulocytes ....................................................................... 112
4.7. Conclusion ...................................