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Gastroenterologie, Hepatologie, Infektionskrankheiten

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Gastroenterologie, Hepatologie, Infektionskrankheiten Aus der Medizinischen Universitätsklinik und Poliklinik Tübingen Abteilung Innere Medizin I Schwerpunkt: Gastroenterologie, Hepatologie, Infektionskrankheiten Überprüfung von Merbromin und Eosin Y als potenzielle Zellregeneration fördernde Substanzen Inaugural-Dissertation zur Erlangung des Doktorgrades der Medizin der Medizinischen Fakultät der Eberhard Karls Universität zu Tübingen vorgelegt von Bajwa, geb. Sindhu, Bariya Ahmed 2019 Dekan: Professor Dr. I. B. Autenrieth 1. Berichterstatter: Professor Dr. N.P. Malek 2. Berichterstatter: Professor Dr. A. Hartkopf Tag der Disputation: 10.07.2019 2 Danksagung An dieser Stelle möchte ich meinen besonderen Dank nachstehenden Personen entgegen bringen, ohne deren Mithilfe die Anfertigung dieser Promotionsschrift niemals zustande gekommen wäre: Mein Dank gilt zunächst Prof. Malek, meinem Doktorvater, für die Betreuung dieser Arbeit, für die vielen Denkanstöße und Ideen, die mir einen kritischen Zugang zu dieser Thematik eröffneten und für die sowohl wissenschaftliche als auch klinische Unterstützung meiner Laufbahn. Ferner möchte ich Dr. Przemyslaw Bozko für die Mitbetreuung an meiner Dissertation und für die freundliche Hilfe danken. Ich danke außerdem Labormitglied Tim Scholta für die stetige Hilfsbereitschaft während meiner wissenschaftlichen Tätigkeit. Tief verbunden und dankbar bin ich meiner Familie und insbesondere meinem Ehemann, Asad Hameed Bajwa, für seine unglaublich hilfreiche Unterstützung in Form von Motivation, als mir diese gefehlt hat, für seine Geduld, und sein Verständnis bei der Anfertigung dieser Doktorarbeit. Mein ganz besonderer Dank aber gilt meinen Eltern, Nasir A. Sindhu und Mahmooda A. Sindhu, die mir meinen bisherigen Lebensweg ermöglichten und denen ich diese Arbeit widme. 3 Inhaltsverzeichnis Danksagung ............................................................................................................... 3 Inhaltsverzeichnis ....................................................................................................... 4 Abkürzungsverzeichnis ............................................................................................... 7 1. Einleitung ............................................................................................................. 9 1.1. Grundlagen der Zellteilungskontrolle ............................................................. 9 1.1.1. Eukaryotischer Zellzyklus im Überblick ................................................... 9 1.1.2. Regulationsmechanismen des Zellzyklus ............................................. 10 1.1.2.1. Cyclin-abhängige Kinasen .............................................................. 10 1.1.2.2. Cyclin-Kinase inhibierende Proteine ............................................... 12 1.1.2.3. Kontrollpunkte des Zellzyklus ......................................................... 15 1.1.3. Cdk2-Aktivität als wichtiger Zellzyklus-Regulationsmechanismus ........ 20 1.1.4. Das Protein p27 als wichtiger Modulator der Cdk2-Aktivität ................. 20 1.2. Regenerative Substanzen ........................................................................... 21 1.2.1. Regenerative Medizin ........................................................................... 21 1.2.1.1. Humane Zellen ............................................................................... 21 1.2.1.2. Biomatrix ........................................................................................ 22 1.2.1.3. Signalstoffe ..................................................................................... 23 1.2.2. Cdk2-aktivierende Substanzen ............................................................. 24 1.2.2.1. Merbromin ...................................................................................... 25 1.2.2.2. Eosin Y ........................................................................................... 27 1.2.3. Vorarbeiten zu Cdk2-aktivierenden Substanzen ................................... 27 1.3. Aufgabenstellung ......................................................................................... 28 2. Material und Methoden ...................................................................................... 30 2.1.1. Material ................................................................................................. 30 2.1.2. Chemikalien .......................................................................................... 30 2.1.3. Antibiotika ............................................................................................. 32 4 2.1.4. Antikörper.............................................................................................. 32 2.1.5. Zelllinien ................................................................................................ 33 2.1.6. Kits ........................................................................................................ 33 2.1.7. shRNA Bibliothek .................................................................................. 33 2.1.8. Verbrauchsmaterialien .......................................................................... 34 2.1.9. Lösungen .............................................................................................. 36 2.2. Methoden .................................................................................................... 40 2.2.1. Kultivieren von adhärenten Zelllinien .................................................... 40 2.2.2. Auszählen von NIH3T3 Zellen mittels Neubauer-Zählkammer ............. 40 2.2.3. Bestimmung der Verdopplungszeit von NIH3T3 Zellen ........................ 41 2.2.4. Untersuchung der Zellvitalität mittels MTT Assay ................................. 42 2.2.5. Untersuchung der Migration mithilfe des Migrations-Assays ................ 42 2.2.6. Zellzyklusanalyse mittels Durchflusszytometrie .................................... 43 2.2.7. Proteincharakterisierung mittels Immunodetektion (Western blot) ........ 44 2.2.7.1. Herstellung der Zelllysate und Messung des Proteingehaltes ........ 44 2.2.7.2. Proteinauftrennung durch die SDS Gelelektrophorese ................... 45 2.2.7.3. Western-Blotting-Methode .............................................................. 47 2.2.7.4. Immunodetektion von Proteinen auf Nitrozellulosemembranen ..... 47 2.2.8. BrdU Analyse mittels Durchflusszytometrie .......................................... 48 2.2.9. Isolierung genomischer DNA aus einer Zellsuspension ....................... 49 2.2.10. shRNA Bibliothek-Screening ................................................................. 49 2.2.10.1. Virusherstellung (Transfektion) ....................................................... 49 2.2.10.2. Transduktion des Virus auf die Zielzelllinie ..................................... 50 2.2.10.3. Durchführung des Cancer 1000 shRNA Screens ........................... 50 3. Ergebnisse ........................................................................................................ 52 3.1. Bestimmung der Verdopplungszeit .............................................................. 52 3.2. Zellviabilitätsanalyse mittels MTT-Assay ..................................................... 53 3.3. Wundverschlussanalyse mittels Migration-Assay ........................................ 55 5 3.4. Zellzyklus-Analyse mittels Durchflusszytometrie ......................................... 59 3.5. Proteinanalyse mittels Western-Blot ............................................................ 64 3.6. Zellteilungsanalyse mittels BrdU-Staining ................................................... 68 3.7. shRNA Screening ........................................................................................ 73 3.7.1. Testreihen für den Versuchsablauf ....................................................... 74 3.7.2. Virus-Transduktion ................................................................................ 75 3.7.3. shRNA Screening-Ergebnisse .............................................................. 76 3.7.3.1. Eosin Y ........................................................................................... 76 3.7.3.2. Merbromin ...................................................................................... 77 4. Diskussion ......................................................................................................... 82 4.1. Untersuchung des Migrationsverhaltens von Zellen unter Merbromin und Eosin Y Behandlung ............................................................................................. 83 4.2. Analyse des Zellzyklus unter Behandlung mit Merbromin und Eosin Y ....... 84 4.2.1. Untersuchung des Zellzyklus anhand der Messung des DNA-Gehalts von Zellen unter Merbromin und Eosin Y Behandlung ...................................... 84 4.2.2. Untersuchung der Veränderung von Proteinmarkern des Zellzyklus unter Merbromin-Behandlung ..................................................................................... 87 4.2.3. Untersuchung der DNA-Synthese-Fähigkeit von Zellen unter Merbromin- Behandlung ....................................................................................................... 90 4.3. Untersuchung der behandelten Zellen mittels shRNA-Screening ................ 93 4.3.1. Eosin Y Screen ..................................................................................... 93 4.3.2. Merbromin-Screen ................................................................................ 95 5. Zusammenfassung .........................................................................................
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