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Aus Dem Institut Für Allgemeine Pathologie Der Medizinischen Fakultät Charité – Universitätsmedizin Berlin Aus dem Institut für allgemeine Pathologie Der Medizinischen Fakultät Charité – Universitätsmedizin Berlin DISSERTATION TP53-abhängige DNA-Reparatur- und Regulationsmechanismen im klassischen Hodgkin-Lymphom und B-Zell-Non-Hodgkin-Lymphomen zur Erlangung des akademischen Grades Doctor medicinae (Dr. med.) vorgelegt der Medizinischen Fakultät Charité – Universitätsmedizin Berlin von Claudia Petereit aus Staßfurt Gutachter/in: 1. Prof. Dr. Michael Hummel 2. PD Dr. Wolfram Klapper 3. Prof. Dr. Matthias Dobbelstein Datum der Promotion: 18.11.2011 ______________________________________________________________________________I 1 Einleitung ..................................................................................................................... 1 1.1 Maligne Lymphome ........................................................................................................ 1 1.1.1 Non-Hodgkin-Lymphome (NHL) ............................................................................... 1 1.1.2 Hodgkin-Lymphom (HL) ............................................................................................ 2 1.1.2.1 Hodgkin- und Reed-Sternberg-Zellen (HRS-Zellen) .............................................. 3 1.1.2.2 Immunphänotyp und Ursprung der HRS-Zellen ..................................................... 4 1.1.2.3 Genetische Aberrationen in HRS-Zellen ................................................................ 5 1.2 Regulation von Zellzyklus, DNA-Reparatur und Apoptose ........................................... 5 1.2.1 Zellzyklus .................................................................................................................... 5 1.2.2 DNA-Reparatur ........................................................................................................... 7 1.2.3 Apoptose ...................................................................................................................... 8 1.3 Tumorsuppressorgen TP53 ............................................................................................. 9 1.3.1 TP53-Struktur .............................................................................................................. 9 1.3.2 Aktivierung und Funktion von TP53 ......................................................................... 10 1.3.3 Regulation von TP53 ................................................................................................. 12 1.3.4 Auswirkungen von Defekten des TP53-Gens und Störungen des P53-Signalwegs in Lymphomen ............................................................................................................... 13 1.4 Aufgabenstellung der Arbeit ......................................................................................... 14 2 Material und Methoden .............................................................................................. 16 2.1 Material ......................................................................................................................... 16 2.1.1 Zelllinien ................................................................................................................... 16 2.1.2 Primer ........................................................................................................................ 17 2.1.3 Reagenzien ................................................................................................................ 17 2.1.3.1 Geräte und Reagenzien für die Behandlung der Zelllinien ................................... 17 2.1.3.2 Reagenzien und Materialien für den Westernblot................................................. 17 2.1.3.3 Puffer ..................................................................................................................... 18 2.1.3.4 Gele ....................................................................................................................... 19 2.1.3.5 Reagenzien und Materialien für die Klonierung ................................................... 19 2.1.3.6 RNA-Isolierung ..................................................................................................... 19 2.1.3.7 Reverse-Transkriptase-Polymerasekettenreaktion (RT-PCR) .............................. 20 2.1.3.8 Reagenzien und Materialien für die Genexpressionsanalyse ................................ 20 2.1.3.8.1 cDNA-Synthese ........................................................................................... 20 2.1.3.8.2 In-vitro-Transkription .................................................................................. 20 2.1.3.8.3 cDNA-, cRNA-Aufreinigung und Fragmentierung ..................................... 20 ______________________________________________________________________________II 2.1.3.8.4 Hybridisierung ............................................................................................. 20 2.1.3.8.5 Färbereagenzien ........................................................................................... 20 2.1.3.8.6 Puffer ........................................................................................................... 21 2.1.3.8.7 Microarrays .................................................................................................. 21 2.1.3.8.8 Software ....................................................................................................... 21 2.1.3.8.9 Affymetrix-Gerätekomponenten ................................................................. 21 2.2 Methoden ....................................................................................................................... 22 2.2.1 Kultur von humanen Zelllinien ................................................................................. 22 2.2.2 Zellbestrahlung .......................................................................................................... 22 2.2.3 Zelllyse und Bestimmung der Zellzahl...................................................................... 23 2.2.4 Bradford-Assay ......................................................................................................... 23 2.2.5 Westernblot ............................................................................................................... 24 2.2.5.1 Gelelektrophorese zur Auftrennung von Proteinen .............................................. 24 2.2.5.2 Immunoblot ........................................................................................................... 25 2.2.5.3 Blocken ................................................................................................................. 25 2.2.5.4 Immunodetektion .................................................................................................. 26 2.2.5.5 Detektion ............................................................................................................... 26 2.2.5.6 Aktin-Kontrolle ..................................................................................................... 26 2.2.6 Isolierung von Gesamt-RNA ..................................................................................... 27 2.2.7 Quantifizierung von Nukleinsäuren (Nanodrop) ....................................................... 27 2.2.8 Reverse-Transkriptase-Polymerasekettenreaktion (RT-PCR) zum Nachweis von TP53 .......................................................................................................................... 28 2.2.9 Agarosegelektrophorese ............................................................................................ 29 2.2.10 Ethidiumbromidfärbung ............................................................................................ 30 2.2.11 Isolierung und Aufreinigung von DNA-Fragmenten ................................................ 30 2.2.12 Klonierung ................................................................................................................. 30 2.2.12.1 Ligation ............................................................................................................ 30 2.2.12.2 3`-A-Überhang ................................................................................................. 31 2.2.12.3 Transformation ................................................................................................. 31 2.2.12.4 Kolonie-PCR .................................................................................................... 32 2.2.13 Sequenzierung ........................................................................................................... 33 2.2.14 Sequenzauswertung ................................................................................................... 33 2.2.15 Qualitätsanalyse der Gesamt-RNA im Bioanalyzer .................................................. 34 2.2.16 Genexpressionsanalyse (GeneChip Array)................................................................ 34 ______________________________________________________________________________III 2.2.16.1 GeneChip® Human Genome U133A 2.0 Array .............................................. 36 2.2.16.2 Herstellung Biotin-markierter Proben für die Affymetrix-Chip-Hybridisierung 36 2.2.16.2.1 Erststrangsynthese der cDNA .................................................................... 36 2.2.16.2.2 Zweitstrangsynthese .................................................................................. 37 2.2.16.2.3 Aufreinigung der Doppelstrang-cDNA ..................................................... 37 2.2.16.2.4 In-vitro-Transkription (IVT).....................................................................
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