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Molekulare Analysen Zur Knochenregeneration Im Alter Und Bei Osteoporose

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Molekulare Analysen Zur Knochenregeneration Im Alter Und Bei Osteoporose Molekulare Analysen zur Knochenregeneration im Alter und bei Osteoporose Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Peggy Benisch geboren in Zeitz Würzburg, 2011 Eingereicht am: Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Thomas Dandekar Gutachter: Prof. Dr. Franz Jakob 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. Würzburg, 04.03.2011 Peggy Benisch Inhaltsverzeichnis 1 Zusammenfassung ........................................................................................................................... 1 2 Summary.......................................................................................................................................... 2 3 Einleitung ......................................................................................................................................... 3 3.1 Knochenhomöostase ............................................................................................................... 3 3.1.1 Knochenumbau ................................................................................................................... 4 3.1.1.1 Knochenabbau ............................................................................................................. 4 3.1.1.2 Knochenaufbau............................................................................................................ 5 3.1.2 Quelle der Regeneration: Mesenchymale Stammzellen (MSC) .......................................... 7 3.1.3 Steuerung des Knochenumbaus .......................................................................................... 8 3.1.3.1 WNT-Signalweg ........................................................................................................... 8 3.1.3.2 BMP-Signalweg ............................................................................................................ 9 3.1.3.3 Wachstumsfaktoren .................................................................................................. 10 3.1.3.4 Mechanotransduktion ............................................................................................... 12 3.1.3.5 Hormonelle Steuerung .............................................................................................. 13 3.2 Alterung ................................................................................................................................. 15 3.2.1 Alterung des Organismus .................................................................................................. 15 3.2.2 Zelluläre Seneszenz ........................................................................................................... 15 3.2.3 Ursachen für Alterung ....................................................................................................... 17 3.2.3.1 Telomere ................................................................................................................... 17 3.2.3.2 DNA- Reparatur ......................................................................................................... 17 3.2.3.3 Oxidativer Stress ........................................................................................................ 18 3.2.3.4 Proliferationsstress und Mitogene ............................................................................ 18 3.2.3.5 Epigenetik .................................................................................................................. 19 3.2.3.6 Genetische Prädisposition ......................................................................................... 20 3.2.4 Immunseneszenz ............................................................................................................... 20 3.2.5 Auswirkung der Alterung auf Knochenhomöostase .......................................................... 22 3.2.5.1 Auswirkungen der Alterung auf MSC ........................................................................ 22 3.3 Osteoporose .......................................................................................................................... 24 3.3.1 Gestörte Knochenhomöostase bei Osteoporose .............................................................. 24 3.3.2 Ursachen für Osteoporose ................................................................................................ 24 3.3.2.1 Alterung ..................................................................................................................... 24 3.3.2.2 Veränderungen im Hormonspiegel ........................................................................... 24 3.3.2.3 Verhalten ................................................................................................................... 25 3.3.2.4 Genetik ...................................................................................................................... 25 3.3.3 MSC und Osteoporose ....................................................................................................... 26 3.4 Ziel der Arbeit ........................................................................................................................ 26 4 Material und Methoden ................................................................................................................ 27 4.1 Material ................................................................................................................................. 27 4.1.1 Geräte ................................................................................................................................ 27 4.1.2 Verbrauchsmaterial ........................................................................................................... 28 4.1.3 Chemikalien/ Reagenzien .................................................................................................. 28 4.1.4 Kits ..................................................................................................................................... 30 4.1.5 Primer ................................................................................................................................ 31 4.1.5.1 Primer für semi-quantitative PCR .............................................................................. 31 4.1.5.2 Sequenzierungsprimer .............................................................................................. 33 4.1.6 Enzyme .............................................................................................................................. 33 4.1.7 Antikörper.......................................................................................................................... 33 4.1.7.1 Primärantikörper ....................................................................................................... 33 4.1.7.2 Sekundärantikörper ................................................................................................... 34 4.1.8 Proteine ............................................................................................................................. 34 4.1.9 Kompetente Bakterien ...................................................................................................... 34 4.1.10 Vektoren ........................................................................................................................ 35 4.1.11 Software und Internet-Seiten ........................................................................................ 35 4.1.1 Lösungen für Molekularbiologie und Proteinbiochemie ................................................... 36 4.1.2 Bakteriennährmedien/-nährboden ................................................................................... 39 4.1.3 Zellkulturmedien ............................................................................................................... 39 4.2 Methoden .............................................................................................................................. 40 4.2.1 Molekularbiologische Methoden ...................................................................................... 40 4.2.1.1 Isolation von zellulärer RNA ...................................................................................... 40 4.2.1.2 Konzentrationsbestimmung von Nukleinsäuren ....................................................... 40 4.2.1.3 Reverse Transkription ................................................................................................ 40 4.2.1.4 Semi-quantitative Polymerase Kettenreaktion (PCR) ............................................... 41 4.2.1.5 Agarosegelelektrophorese ........................................................................................ 42 4.2.1.6 Densitometrie ............................................................................................................ 42 4.2.1.7 Agarosegel-Eluation................................................................................................... 42 4.2.1.8 DNA-Sequenzierung .................................................................................................
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