Characterization of human mesenchymal stem cells by the appearance of integrins and functional analysis of collagen I-binding integrins [Elektronische Ressource] / vorgelegt von Tzvetan Popov

Popov, Tzvetan

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 23
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Aus der Chirurgisches Forschungslabor,
Chirurgischen Klinik und Poliklinik – Innenstadt,
der Ludwig-Maximilians-Universität zu München
Direktor: Prof. Dr. med. Wolf Mutschler





Characterization of human mesenchymal stem cells by the appearance of
integrins and functional analysis of collagen I-binding integrins





Dissertation
zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München





vorgelegt von
Tzvetan Popov

aus
Plovdiv, Bulgaria

Jahr
2011
2
Mit Genehmigung der Medizinischen Fakultät
der Universität München






Berichterstatter: Prof. Dr. med. Matthias Schieker
Prof. Magdalena Götz
Mitberichterstatter: Prof. Dr. Dr. Ultich Welsch Dr. Eugen Faist

Mitbetreuung durch den
promovierten Mitarbeiter: Dr. rer. nat. Denitsa Docheva
Dekan: Prof. Dr. med. Dr. h.c. M.Reiser, FACR, FRCR

Tag der mündlichen Prüfung: 22.02.2011




Content table 3
CONTENT TABLE
1. Introduction ....................................................................................................................................................... 5
1.1. Human mesenchymal stem cells (hMSC)......................................................................................................... 5
1.1.1. Definition and sources................................................................................................................................... 5
1.1.2. Criteria and markers ...................................................................................................................................... 6
1.1.3. BM cell niche ................................................................................................................................................ 7
1.2. Cell surface receptors................................. 12
1.2.1. Cell-cell receptors........................................................................................................................................ 12
1.2.2. Cell-matrix receptors................................................................................................................................... 13
1.2.3. Integrins....................................................................................................................................................... 14
1.2.3.1. Focal adhesions and integrin signaling..................................................................................................... 16
1.2.3.2. Importance of integrin signaling............................................................................................................... 18
1.2.3.3. In vivo functions of integrins.................................................................................................................... 21
1.3. Integrin expression in hMSC.......................................................................................................................... 23
1.4. Methods of studying gene function in human cells ........................................................................................ 24
1.4.1. History of RNAi .......................................................................................................................................... 25
1.4.2. Mechanism of RNA interference................................................................................................................. 25
1.4.3. Advantages and disadvantages of the RNAi technology............................................................................. 27
1.4.4. Usefulness of RNAi technology....................... 29
2. Aim and goals of the thesis ............................................................................................................................. 31
3. Material and Methods..................................................................................................................................... 32
3.1. Human primary cells and cell lines ................................................................................................................ 32
3.2. Culture conditions.................................. 33
3.2.1. Passaging and counting ............................................................................................................................... 34
3.2.2. Cryopreservation................................. 34
3.3. ShRNA sequences .......................................................................................................................................... 34
3.4. Bacterial cloning............................................................................................................................................. 35
3.4.1. Bacterial strains ....................................................................................................... 35
3.4.2. Bacterial culture media................................................................................................................................ 35
3.4.3. Plasmids ...................................................................................................................................................... 36
3.4.4. Ligation and recombination reactions ......................................................................................................... 36
3.4.5. Isolation of plasmid DNA (pDNA) ............................................................................................................. 37
3.5. Virus production............................................................................................................................................. 37
3.6. RNA and copy DNA (cDNA) preparation ..................................................................................................... 38
3.7. RT-PCR.......................................................................................................................................................... 39
3.8. Light Cycler (LC)-PCR .................................................................................................................................. 39
3.9. Cytochemistry ................................................................................................................................................ 40
3.10. Western blotting ........................................................................................................................................... 41
3.11. Cell adhesion assay....................................................................................................................................... 42
3.12. Proliferation assays 43 Content table 4
3.13. Time lapse experiments................................................................................................................................ 43
3.14. Osteogenic differentiation ............................................................................................... 44
3.15. Apoptosis analysis........................................................................................................................................ 45
3.16. Interferon stimulation................................ 45
3.17. Microscopy................................................................................................................................................... 45
3.18. Computer programs and web links............................................................................................................... 46
3.19. Statistics........................................ 46
4. Results .............................................................................................................................................................. 47
4.1. Characterization of hMSC.............................................................................................................................. 47
4.1.1. Morphological appearance and growth capacity of hMSC.......................................................................... 47
4.1.2. HMSC affinity towards different ECM proteins ......................................................................................... 48
4.1.3. Influence of different ECM proteins of hMSC proliferation capability ...................................................... 48
4.1.4. Integrin expression in hMSC....................................................................................................................... 49
4.1.4.1. Expression of ColI-binding integrin in hMSC.......................................................................................... 50
4.1.5. Integrin expression changes upon osteogenic stimulation........................................................................... 51
4.1.6. Changes in a1, a2 and a11 integrin expression upon osteogenic stimulation. ............................................. 52
4.2. Establishment of a stable knockdown system for ColI-binding integrins....................................................... 54
4.2.1. Cloning of lentiviral constructs for expression of a1, a2 and a11 shRNA................................................... 54
4.2.2. Knockdown of integrins a1, a2 and a11 in hMSC............ 56
4.2.3. Morphologica