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Identifizierung Und Charakterisierung Von Myokinen Und Adipokinen Mit Bedeutung Für Die Körperzusammensetzung Beim Rind

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Identifizierung Und Charakterisierung Von Myokinen Und Adipokinen Mit Bedeutung Für Die Körperzusammensetzung Beim Rind Leibniz-Institut für Nutztierbiologie (FBN) Institut für Muskelbiologie und Wachstum Identifizierung und Charakterisierung von Myokinen und Adipokinen mit Bedeutung für die Körperzusammensetzung beim Rind Dissertation zur Erlangung des Doktorgrades der Ernährungswissenschaften (Dr. troph.) der Naturwissenschaftlichen Fakultät III Agrar- und Ernährungswissenschaften, Geowissenschaften und Informatik der Martin-Luther-Universität Halle-Wittenberg vorgelegt von Lisa Schering geboren am 24.03.1988 in Lutherstadt Wittenberg Dekan: Prof. Dr. Olaf Christen 1. Gutachter: Prof. Dr. Steffen Maak 2. Gutachterin: Prof. Dr. Gabriele Stangl 3. Gutachterin: Prof. Dr. Susanne Klaus Tag der Verteidigung: 12.12.2016 Inhaltsverzeichnis Abkürzungsverzeichnis ....................................................................................................... IV Abbildungsverzeichnis ........................................................................................................ VI Tabellenverzeichnis ........................................................................................................... VIII 1 Einführung ....................................................................................................................... 1 2 Literaturübersicht ........................................................................................................... 3 2.1 Fettgewebe als endokrines Organ .................................................................... 3 2.2 Skelettmuskelgewebe als endokrines Organ .................................................... 6 2.3 Wechselwirkungen zwischen Skelettmuskel- und Fettgewebe ......................... 7 2.3.1 Adipokine .......................................................................................................... 9 2.3.2 Myokine ........................................................................................................... 11 2.3.3 Adipo-Myokine ................................................................................................ 14 2.4 Zielsetzung ...................................................................................................... 16 3 Material und Methoden ................................................................................................. 17 3.1 Probenmaterial ................................................................................................ 17 3.1.1 Rinder aus einer experimentellen F2-Kreuzung .............................................. 17 3.1.2 Rinderrassen mit differenter IMF-Akkumulation .............................................. 17 3.1.3 Gewebe für molekularbiologische und proteinbiochemische Analysen .......... 17 3.1.4 Zelllinie ............................................................................................................ 18 3.1.5 Primäre bovine Zellen ..................................................................................... 18 3.1.5.1 Isolierung von stromavaskulären Zellen .......................................................... 18 3.1.5.2 Isolierung von bovinen Satellitenzellen ........................................................... 19 3.1.5.3 Gewinnung von Fettgewebe für Explant-Kulturen ........................................... 19 3.1.6 Chemikalien, Puffer, Verbrauchsmaterialien, Software und Geräte ................ 20 3.2 Methoden ........................................................................................................ 20 3.2.1 Datenanalyse .................................................................................................. 20 3.2.2 Lasermikrodissektion ...................................................................................... 21 3.2.3 Molekularbiologische Analysen ....................................................................... 22 3.2.3.1 RNA-Isolierung ................................................................................................ 22 3.2.3.2 Konzentrationsmessung der RNA ................................................................... 22 3.2.3.3 Isolierung von genomischer DNA .................................................................... 22 3.2.3.4 Proteinisolierung ............................................................................................. 23 3.2.3.5 Proteinfraktionierung ....................................................................................... 23 3.2.3.6 Reverse Transkription (cDNA-Synthese) ........................................................ 24 3.2.3.7 Primerdesign ................................................................................................... 24 3.2.3.8 PCR und Agarosegelelektrophorese für Primertests ...................................... 26 3.2.3.9 Quantitative Echtzeit-PCR (RT-qPCR)............................................................ 27 I 3.2.3.10 Strukturelle Analyse des THRSP-Locus ........................................................ 29 3.2.4 Zellkultur .......................................................................................................... 30 3.2.4.1 Kultivierung und Differenzierung von 3T3-L1-Zellen ....................................... 30 3.2.4.2 Kultivierung und Differenzierung von primären bovinen Präadipozyten.......... 31 3.2.4.3 Ölrot-Färbung .................................................................................................. 31 3.2.4.4 Fettexplantkultur aus SCF ............................................................................... 32 3.2.4.5 Kultivierung und Differenzierung von bovinen Satellitenzellen ....................... 32 3.2.5 Proteinbiochemische Methoden ...................................................................... 33 3.2.5.1 Immunhistochemie .......................................................................................... 33 3.2.5.1.1 3T3-L1-Zellen ............................................................................................ 33 3.2.5.1.2 Gewebeschnitte der bovinen Leber, des Skelettmuskels und des subkutanen Fettgewebes .......................................................................... 34 3.2.5.1.3 Kapillarfärbung vom bovinen Muskelquerschnitt ...................................... 35 3.2.5.1.4 Primäre bovine Muskelzellen .................................................................... 36 3.2.5.2 Western Blot .................................................................................................... 36 4 Ergebnisse ..................................................................................................................... 41 4.1 Vergleichende Analyse von Transkriptom-, Proteom- und Sekretomdaten der Spezies Rind, Mensch und Maus .................................................................... 41 4.1.1 Evaluierung different exprimierter Transkripte im Fettgewebe und Skelettmuskel .................................................................................................. 41 4.1.2 Identifizierung von Adipokinen und Myokinen als Teilmenge der Transkripte mit High-Score ................................................................................................ 41 4.1.3 Identifizierung von Adipo-Myokinen ................................................................ 42 4.1.4 Netzwerk-Analyse der Gene mit High-Score .................................................. 44 4.2 Vergleichende Expressionsanalysen von ausgewählten Kandidatengenen im Skelettmuskel- und Fettgewebe von Bullen der SEGFAM-Population ............ 49 4.2.1 Analyse von THBS4 beim Rind ....................................................................... 50 4.2.2 Analyse von THRSP beim Rind ...................................................................... 54 4.2.2.1 mRNA-Expression von THRSP in verschiedenen bovinen Geweben............. 55 4.2.2.2 Analyse und vergleichende Sequenzierung des THRSP-Locus ..................... 56 4.2.2.3 Genexpression von THRSP auf mRNA- und Proteinebene in verschiedenen Zelltypen .......................................................................................................... 56 4.2.2.4 mRNA-Expression während der adipogenen Differenzierung......................... 62 4.2.2.5 Proteinlokalisation in der 3T3-L1-Zelllinie ....................................................... 64 4.3 Etablierung von primären bovinen Zellkulturmodellen für funktionelle Untersuchungen .............................................................................................. 66 II 4.3.1 Kultivierung und adipogene Differenzierung von primären bovinen stromavaskulären Zellen ................................................................................. 66 4.3.2 Relative mRNA-Expression von Genen in bovinen Fettexplants .................... 68 4.3.3 Kultivierung von primären bovinen Myoblasten .............................................. 70 5 Diskussion ..................................................................................................................... 73 5.1 Speziesübergreifende Genexpressionsdaten-Analyse ................................... 73 5.2 Initiale Charakterisierung ausgewählter, putativer Adipokine und Myokine beim Rind ........................................................................................................ 80 5.3 Etablierung von primären bovinen Zellkulturmodellen zur funktionellen Untersuchung von potentiellen Adipokinen und Myokinen ............................. 88 5.4 Fazit ................................................................................................................ 93 6 Zusammenfassung ......................................................................................................
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