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Und Endothelzellen Aus dem Universitätsklinikum Münster Klinik und Poliklinik für Mund-, und Kiefer-Gesichtschirurgie des Zentrums für Zahn-, Mund- und Kieferheilkunde - Direktor: Univ.- Prof. Dr. Dr. Dr.h.c. U. Joos - ____________________________________________________________________ Genexpressionsanalyse der kokulturellen Verhältnisse zwischen Tumor- und Endothelzellen INAUGURAL-DISSERTATION zur Erlangung des doctor medicinae dentium der Medizinischen Fakultät der Westfälischen Wilhelms-Universität Münster vorgelegt von Krause, Thomas Fritz Rudi Gerhard aus Mühlhausen/Thüringen 2006 Gedruckt mit Genehmigung der Medizinischen Fakultät der Westfälischen Wilhelms-Universität Münster Dekan: Univ.-Prof. Dr. Heribert Jürgens 1. Berichterstatter: Priv.-Doz. Dr. Dr. J. Kleinheinz 2. Berichterstatter: Prof. Dr. P.Scheutzel Tag der mündlichen Prüfung: 30.10.20006 Aus dem Universitätsklinikum Münster Klinik und Poliklinik für Mund-, und Kiefer-Gesichtschirurgie des Zentrums für Zahn-, Mund- und Kieferheilkunde - Direktor: Univ.- Prof. Dr. Dr. Dr.h.c. U. Joos - Zusammenfassung Genexpressionsanalyse der kokulturellen Verhältnisse zwischen Tumor- und Endothelzellen Thomas Krause Die Behandlung von neoplastischen Gewebsveränderungen des Kopf-Halsbereiches hat sich in den letzten Jahrzehnten stetig verbessert. Gerade das Plattenepithelkarzinom, als häufigstes Malignom dieser Region, zeigt jedoch eine relativ schlechte Gesamtprognose. Diese Tatsache kann durch einen stärkeren Bezug der Diagnostik und Therapie auf die eigentliche Tumorbiologie verbessert werden. Die dann mögliche exaktere Prognose oder individuellere Therapie fordert jedoch ein absolutes Verständnis der zugrunde liegenden Mechanismen. Die vorliegende Arbeit verfolgt die Zielstellung die transkriptionelle Aktivität von Plattenepithelkarzinomzellen in Abhängigkeit der An- oder Abwesenheit von Endothelzellen mittels einer umfassenden Genexpressionsanalyse zu untersuchen. Der Genexpressionsvergleich zwischen Tumorkultur und Tumor-Endothelzellkokultur zeigte deutliche Expressionsunterschiede, so wurde der Einfluss des tumorumgebenden Gewebes im Verlauf der Arbeit immer deutlicher. Ebenso zeigt sich die Bedeutung von komplexen in vitro Kokulturen und deren umfassende Genexpressionsanalyse zur Simulation von in vivo Vorgängen. Tumorkulturell wurden Interleukine- (IL1A, IL8), Chemokine (CXCL1, CCL20, CXCL3 und CX3CL1), die ‚Tumor Nekrosis Factors’ TNFAIP2, TNFRSF6 und TRAF4, und ebenso die angiogenen Faktoren ECGF und Angiogenin erhöht exprimiert. Gegensätzlich dazu zeigte die Tumor-Endothelzellkokultur eine erhöhte Expression von PLAUR, ESDN, GFI-1, ID-3, CCND2 und zudem wurde der MAP-Kinase-Stoffwechselweg positiv beeinflusst (CACNG6, DUSP10, RASGRP1, TNFRSF6, CXCL1). In der Tumorkultur ist es zu einer Expression von positiv angiogen-proliferativen Faktoren gekommen. Die expressionellen Charakteristika der Tumorkultur zeigen einen sehr aktiven Tumorstoffwechsel, der auf angiogenetische Induktion, Zellproliferation, Wachstum und Apoptoseinhibition hinzielt. Kokulturell steht weniger die Expression von proangiogenetischen Faktoren im Vordergrund als die Expression von Faktoren der Invasion, Migration und Metastasierung. Der Tumorzelle genügt demnach die Simulation eines Gefäßplexus durch die endotheliale Anwesenheit innerhalb einer in-vitro Tumor-Endothelzellkokultur um von einem proangiogenen Expressionsschema hin zum invasiven und metastasierenden Expressionsmuster zu gelangen. Tag der mündlichen Prüfung: 30.10.2006 Diese Dissertation ist in Dankbarkeit meinen Eltern gewidmet. Inhaltsverzeichnis Einleitung......................................................................................................................................- 1 - 1 Wissenschaftlicher Hintergrund............................................................................................- 2 - 1.1 Die Endothelzelle........................................................................................................- 2 - 1.1.1 Zytologie und Histologie.....................................................................................- 2 - 1.1.2 Beteiligung der Endothelzelle an der Koagulation und Antikoagulation................- 3 - 1.1.3 Endothelzellen und Immunreaktionen.................................................................- 4 - 1.1.4 Angiogenese......................................................................................................- 5 - 1.2 Die Tumorzelle............................................................................................................- 9 - 1.2.1 Ätiologie der Mundhöhlenkarzinome...................................................................- 9 - 1.2.2 Feldkanzerogenisierung ...................................................................................- 10 - 1.2.3 „Multi-Step-Theory“..........................................................................................- 10 - 1.2.4 Epidemiologie..................................................................................................- 11 - 1.2.5 Tumorangiogenese..........................................................................................- 11 - 1.2.6 Invasion und Metastasierung ...........................................................................- 13 - 1.2.7 Tumordiagnostik..............................................................................................- 14 - 1.2.8 Tumortherapie.................................................................................................- 15 - 1.3 Genexpressionsbeobachtung ....................................................................................- 17 - 1.3.1 Einleitung ........................................................................................................- 17 - 1.3.2 Methodik der „GeneChips“-Affymetrix...............................................................- 18 - 1.3.3 Methodik der „TaqMans“-Applied Biosystems...................................................- 19 - 1.3.4 Photolithographie – GeneChips Technologie - Affymetrix .................................- 19 - 1.3.5 Chemilumineszenz- TaqMan Technologie – Applied Biosystems ......................- 20 - 1.4 Ein Vergleich von Affymetrix und Applied Biosystems ...............................................- 24 - 2 Problemstellung.................................................................................................................- 26 - 3 Material und Methode.........................................................................................................- 27 - 3.1 Isolierung von Endothelzellen aus humaner Nabelschnurvene...................................- 27 - 3.2 Passagieren- u. Aussaat v. Endothelzellen humaner Nabelschnurvenen....................- 28 - 3.3 CAL27 – experimentelle Tumorzelllinie –...................................................................- 28 - 3.4 Herstellung einer Kokultur aus CAL27 Tumorzellen und Endothelzellen. ....................- 29 - 3.5 Ablösung der Zellen und RNA Extraktion für die Genchip-Analyse.............................- 29 - 3.6 RNA Isolierung aus Tumorgeweben ..........................................................................- 30 - 3.7 Bestimmung der Konzentration und Reinheit der DNA und RNA................................- 30 - 3.8 „Human Genom Survey Microarrays“ ........................................................................- 30 - 3.9 Statistische Auswertung............................................................................................- 32 - 3.9.1 t-Test-Mittelwertsvergleiche..............................................................................- 32 - 3.9.2 F-Test - ...........................................................................................................- 33 - 3.9.3 FC – der Fold Change......................................................................................- 33 - 3.10 Auswertung der Ergebnisse der Genexpressionsuntersuchung ..................................- 33 - 4 Ergebnisse.........................................................................................................................- 35 - 4.1 Einleitung .................................................................................................................- 35 - 4.2 Tumorreinkulturell erhöhte Transkription ...................................................................- 36 - 4.2.1 TNF-assoziierte Moleküle.................................................................................- 36 - 4.2.2 Chemokine ......................................................................................................- 36 - 4.2.3 Cytochrome und Serpine .................................................................................- 37 - 4.2.4 Interleukine......................................................................................................- 38 - 4.2.5 Interferon-assoziierte Moleküle.........................................................................- 39 - 4.2.6 angiogenetische Faktoren ................................................................................- 39 - 4.2.7 Invasion und Metastasierung ...........................................................................- 40 - 4.2.8 Transkriptionelle Faktoren................................................................................- 41 - 4.2.9 Homeobox und nukleäre Faktoren....................................................................- 42 - 4.2.10 Endo- und Exocytose.......................................................................................-
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