Transdifferentiation of human mesenchymal stem cells [Elektronische Ressource] / vorgelegt von Tatjana Schilling

Tati

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 18
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Transdifferentiation of
Human Mesenchymal Stem Cells



Dissertation zur Erlangung des naturwissenschaftlichen
Doktorgrades der Julius-Maximilians-Universität Würzburg



vorgelegt von
Tatjana Schilling

aus
San Miguel de Tucuman, Argentinien


Würzburg, 2007























Eingereicht am:


Mitglieder der Promotionskommission:

Vorsitzender: Prof. Dr. Martin J. Müller
Gutachter: PD Dr. Norbert Schütze
Gutachter: Prof. Dr. Georg Krohne


Tag des Promotionskolloquiums:


Doktorurkunde ausgehändigt am:

























Hiermit erkläre ich ehrenwörtlich, dass ich die vorliegende Dissertation selbstständig
angefertigt und keine anderen als die von mir angegebenen Hilfsmittel und Quellen
verwendet habe. Des Weiteren erkläre ich, dass diese Arbeit weder in gleicher noch in
ähnlicher Form in einem Prüfungsverfahren vorgelegen hat und ich noch keinen
Promotionsversuch unternommen habe.

Gerbrunn, 4. Mai 2007




Tatjana Schilling Table of contents i
Table of contents

1 Summary ........................................................................................................................ 1
1.1 Summary.................................................................................................................... 1
1.2 Zusammenfassung..................................................................................................... 2
2 Introduction.................................................................................................................... 4
2.1 Osteoporosis and the fatty degeneration of the bone marrow..................................... 4
2.2 Adipose and bone tissue ............................................................................................ 4
2.2.1 Adipose tissue...................................................................................................... 4
2.2.2 Bone tissue........................................................................................................... 5
2.2.3 Bone remodelling.................................................................................................. 7
2.3 Mesenchymal stem cells ............................................................................................ 8
2.3.1 Multilineage potential............................................................................................ 8
2.3.2 Adipogenesis........................................................................................................ 9
2.3.3 Osteogenesis ..................................................................................................... 11
2.3.4 Inverse relationship between adipogenic and osteogenic differentiation............. 14
2.4 Transdifferentiation................................................................................................... 15
2.5 Objective of this study .............................................................................................. 17
3 Materials and methods ................................................................................................ 18
3.1 Materials .................................................................................................................. 18
3.1.1 Consumables ..................................................................................................... 18
3.1.2 Chemicals and reagents..................................................................................... 18
3.1.3 Primers............................................................................................................... 21
3.1.4 Recombinant proteins......................................................................................... 22
3.1.5 Antibodies .......................................................................................................... 23
3.1.6 Enzymes ............................................................................................................ 23
3.1.7 Buffers and other solutions................................................................................. 23
3.1.8 Cell culture media and additives......................................................................... 27
3.1.9 Kits ..................................................................................................................... 28
3.1.10 Equipment .......................................................................................................... 28
3.1.11 Software and online sources .............................................................................. 30
3.2 Methods ................................................................................................................... 30
3.2.1 Isolation of human mesenchymal stem cells....................................................... 30
3.2.2 Cell culture of human mesenchymal stem cells .................................................. 31
3.2.2.1 Culture and propagation ............................................................................ 31
3.2.2.2 Differentiation............................................................................................. 31
3.2.2.3 Transdifferentiation .................................................................................... 32 Table of contents ii
3.2.2.4 Proliferation assay ..................................................................................... 32
3.2.2.5 Application of recombinant proteins in the cell culture................................ 32
3.2.3 Methods in molecular biology ............................................................................. 34
3.2.3.1 Isolation of cellular RNA............................................................................. 34
3.2.3.2 Reverse transcriptase polymerase chain reaction (RT-PCR) ..................... 34
3.2.3.3 Agarose gel electrophoresis....................................................................... 35
3.2.3.4 DNA sequencing........................................................................................ 35
3.2.3.5 cDNA microarray analysis.......................................................................... 36
3.2.4 Methods in protein analysis ................................................................................ 37
3.2.4.1 Preparation of intracellular protein ............................................................. 37
3.2.4.2 Protein precipitation in culture supernatants .............................................. 38
3.2.4.3 Determination of protein concentration....................................................... 38
3.2.4.4 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .............................. 38
3.2.4.5 Western blotting......................................................................................... 40
3.2.5 Cytochemical methods ....................................................................................... 40
3.2.5.1 Alizarin red S staining ................................................................................ 40
3.2.5.2 Alkaline phosphatase (ALP) staining.......................................................... 41
3.2.5.3 Oilred O staining ........................................................................................ 41
4 Results.......................................................................................................................... 42
4.1 Adipogenic and osteogenic differentiation potential of human mesenchymal stem
cells.......................................................................................................................... 42
4.2 Adipogenic and osteogenic transdifferentiation potential of human mesenchymal stem
cells.......................................................................................................................... 44
4.2.1 Adipogenic transdifferentiation............................................................................ 45
4.2.2 Osteogenic transdifferentiation ........................................................................... 47
4.3 Affymetrix microarray analyses of adipogenic and osteogenic transdifferentiation.... 48
4.3.1 Regulation of gene products shortly after initiation of transdifferentiation............ 48
4.3.1.1 Affymetrix GeneChip HG-U 133A............................................................... 48
4.3.1.2 Affymetrix GeneChip HG-U 133 Plus 2.0 ................................................... 49
4.3.2 Re-evaluation of microarray data by semi-quantitative RT-PCR ......................... 52
4.3.3 Gene ontology analyses ..................................................................................... 58
4.3.4 Functional grouping of differentially regulated genes.......................................... 63
4.3.5 Development of a novel bioinformatic scoring scheme for interpretation of
microarray results.......................................................................