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Diplomarbeit DIPLOMARBEIT Titel der Diplomarbeit „The influence of free fatty acids on the development of liver inflammation“ Verfasser Mario Kuttke, B.Sc. angestrebter akademischer Grad Magister der Naturwissenschaften (Mag.rer.nat.) Wien, 2012 Studienkennzahl lt. Studienblatt: A 490 Studienrichtung lt. Studienblatt: Diplomstudium Molekulare Biologie Betreuerin / Betreuer: A.o.Univ.-Prof.Dipl.-Ing.Dr. Marcela Hermann Danksagung Zuerst möchte ich mich bei a.o.Univ.-Prof. Dipl.-Ing. Dr. Marcela Hermann für die Betreuung meiner Diplomarbeit bedanken. Besonderer Dank gilt a.o.Univ-Prof. Dr. Bettina Grasl-Kraupp für die fachliche Unterstützung und Betreuung während der praktischen Durchführung der Arbeit. Weiters bedanke ich mich bei Sandra Sagmeister, Therese Böhm, Nora Bintner, Waltraud Schrottmaier, Melanie Pichlbauer, Marzieh Nejabat, Teresa Riegler, Bettina Wingelhofer und Christiane Maier für die ausgezeichnete Zusammenarbeit im Labor und die Unterstützung in allen Lebenslagen. Birgit Mir-Karner, Helga Koudelka und Krystyna Bukowska danke ich für ihre Hilfsbereitschaft und für die kollegiale Zusammenarbeit. Mein größter Dank gilt meinen Eltern, Ursula und Heinz, und meiner Großmutter, Theresia, die mir mein Studium ermöglicht und mich immer unterstützt haben, sowie meinem Bruder, Alex, der in allen Lebenslagen für mich da ist. Table of Contents TABLE OF CONTENTS INTRODUCTION ............................................................................................................................................. 4 HEPATOCELLULAR CARCINOMA (HCC) ......................................................................................................................... 4 Hepatocarcinogenesis .................................................................................................................................... 4 OBESITY ................................................................................................................................................................. 7 NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD) ........................................................................................................... 8 Non-alcoholic steatohepatitis (NASH) ............................................................................................................ 9 FREE FATTY ACIDS (FFAS) ........................................................................................................................................ 10 Effects of impaired metabolism of FFAs ....................................................................................................... 11 Types and effects of free fatty acids ............................................................................................................. 11 Free fatty acid receptors ............................................................................................................................... 13 INFLAMMATION AND INFLAMMATION ASSOCIATED FACTORS ........................................................................................... 17 TNFα ............................................................................................................................................................. 18 COX-2 ............................................................................................................................................................ 19 iNOS .............................................................................................................................................................. 19 IL-6 ................................................................................................................................................................ 19 ADAM17/TACE ............................................................................................................................................. 20 AIMS OF THE THESIS .................................................................................................................................... 22 MATERIALS AND METHODS ......................................................................................................................... 23 MATERIALS ........................................................................................................................................................... 23 BUFFERS AND SOLUTIONS ........................................................................................................................................ 26 MEDIA................................................................................................................................................................. 27 CELL CULTURE ....................................................................................................................................................... 28 Animals and in vivo treatment ..................................................................................................................... 28 Collagenase perfusion of the rat liver ........................................................................................................... 28 Purification of primary rat non-parenchymal cells (NPCs) ........................................................................... 29 Purification of primary rat hepatocytes ....................................................................................................... 29 Determination of cell viability ...................................................................................................................... 29 Culture of primary rat non-parenchymal cells .............................................................................................. 29 Culture of primary rat hepatocytes .............................................................................................................. 29 Culture of cell lines ....................................................................................................................................... 29 Treatment of cells with free fatty acids ........................................................................................................ 30 Treatment of cells with Sulfosuccinimidyl oleate sodium (SSO) ................................................................... 30 Cytotoxicity assay ......................................................................................................................................... 30 DETECTION OF MRNA ............................................................................................................................................ 31 RNA isolation using the TriFast reagent ....................................................................................................... 31 RNA isolation using miRNeasy Mini kit (Qiagen) .......................................................................................... 31 Reverse transcription PCR ............................................................................................................................ 31 Polymerase chain reaction (PCR) .................................................................................................................. 32 Quantitative real-time PCR (qRT-PCR) .......................................................................................................... 32 Agarose-gel electrophoresis ......................................................................................................................... 33 Bioanalyzer ................................................................................................................................................... 33 RNA-microarray Array Transcriptome Analysis ............................................................................................ 33 DETECTION OF PROTEIN .......................................................................................................................................... 33 Protein isolation ........................................................................................................................................... 33 Determination of protein concentrations ..................................................................................................... 34 SDS-PAGe ...................................................................................................................................................... 34 Transfer ........................................................................................................................................................ 34 1 Table of Contents Immunodetection ......................................................................................................................................... 34 Enzyme-linked immunosorbent assay (ELISA) .............................................................................................. 35 EXTRACTION OF SERUM FROM RAT BLOOD .................................................................................................................. 35 DETECTION OF FREE FATTY ACIDS .............................................................................................................................. 36 ADAM17/TACE ACTIVITY ASSAY ............................................................................................................................. 36 THP1 ............................................................................................................................................................
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