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Direktor Univ.- Prof. Dr. H. Brinkmeier) Der Universitätsmedizin Der Ernst-Moritz-Arndt-Universität Greifswald

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Direktor Univ.- Prof. Dr. H. Brinkmeier) Der Universitätsmedizin Der Ernst-Moritz-Arndt-Universität Greifswald Aus dem Institut für Pathophysiologie (Direktor Univ.- Prof. Dr. H. Brinkmeier) der Universitätsmedizin der Ernst-Moritz-Arndt-Universität Greifswald "Expression und Lokalisation der Kationenkanäle TRPC3, TRPC6 und TRPV5 in verschiedenen Geweben der Maus" Inaugural - Dissertation zur Erlangung des akademischen Grades Doktor der Medizin (Dr. med.) der Universitätsmedizin der Ernst-Moritz-Arndt-Universität Greifswald 2013 vorgelegt von: Frederike Bisping geb. am: 04.09.1982 in: Winsen/Luhe Dekan: Prof. Dr. med. dent. Reiner Biffar 1. Gutachter: Prof. Dr. Heinrich Brinkmeier, Institut für Pathophysiologie, Universität Greifswald 2. Gutachter: Prof. Dr. med. Antje Bornemann, Institut für Pathologie und Neuropathologie, Universität Tübingen Ort, Raum: Ernst-Moritz-Arndt-Universität Greifswald, Hörsaal Pathologie Tag der Disputation: 06.12.2013 Inhaltsverzeichnis 1. Einleitung ......................................................................................................... 3 1.1 Die TRP Familie von Kationenkanälen ........................................................................... 3 1.2 Aufgaben und Bedeutung der TRP Kanäle ...................................................................... 7 1.3 Krankheiten ...................................................................................................................... 8 1.4. Tiermodelle ................................................................................................................... 11 1.5 Zielstellung der Arbeit ................................................................................................... 12 2. Material und Methoden ................................................................................ 13 2.1 Material .......................................................................................................................... 13 2.1.1 Mausstamm ................................................................................................................. 13 2.1.2 Chemikalien und Enzyme ........................................................................................... 13 2.1.3 Antikörper ................................................................................................................... 15 2.1.4 Enzyme und Primer ..................................................................................................... 15 2.1.5 Kits für die Immunhistochemie und in-situ Hybridisierung ....................................... 16 2.1.6 Plasmid ........................................................................................................................ 17 2.1.7 Verwendete Lösungen ................................................................................................. 17 2.1.8 Geräte .......................................................................................................................... 19 2.2 Methoden ........................................................................................................................ 20 2.2.1 Präparation der Mäusegewebe und Herstellung der Schnitte ...................................... 20 2.2.2 DIG-RNA-Sonden Synthese ....................................................................................... 20 2.2.3 in-situ Hybridisierung ................................................................................................. 21 2.2.4 Indirekte Immunhistochemie ....................................................................................... 23 2.2.5 Indirekte Immunfluoreszenz ....................................................................................... 24 2.2.6 Real-Time TaqMan® Polymerase Kettenreaktion ...................................................... 25 3. Ergebnisse ...................................................................................................... 27 3.1 Quantitative RT-PCR für TRPC3, TRPC6 und TRPV5 ................................................ 27 3.2 In-situ Lokalisation von TRPC3, TRPC6 und TRPV5 .................................................. 30 3.2.1 Etablierung der in-situ Sonden .................................................................................... 31 3.2.2 Kontrollen und Korrespondenzgewebe ....................................................................... 31 3.2.3 In-situ Lokalisation im Gehirn .................................................................................... 35 3.2.4 In-situ Lokalisation in der Lunge ................................................................................ 37 1 3.2.5 In-situ Lokalisation im Herzmuskel und der Aorta ..................................................... 38 3.2.6 In-situ Lokalisation in der Leber ................................................................................. 39 3.2.7 In-situ Lokalisation in der Milz ................................................................................... 39 3.2.8 In-situ Lokalisation im Dünndarm .............................................................................. 40 3.2.9 In-situ Lokalisation in der Niere ................................................................................. 41 3.2.10 In-situ Lokalisation im Pankreas ............................................................................... 42 3.2.11 In-situ Lokalisation in der Skelettmuskulatur ........................................................... 44 3.2.12 In-situ Lokalisation in den männlichen Fortpflanzungsorganen ............................... 44 3.2.13 In-situ Lokalisation in den weiblichen Fortpflanzungsorganen ................................ 45 3.2.14 Zusammenfassung der Ergebnisse ............................................................................ 47 4. Diskussion ....................................................................................................... 49 5. Zusammenfassung ......................................................................................... 55 6. Literaturverzeichnis ...................................................................................... 57 7. Anhang............................................................................................................ 60 “Tissue-specific expression of TRP channel genes in the mouse and its variation in three different mouse strains.” von Kunert-Keil, Bisping, Krüger, Brinkmeier, BMC Genomics 2006, 7:159 "Transient receptor potential cation channels in normal and dystrophic mdx muscle.” Krüger, Kunert-Keil, Bisping, Brinkmeier, Neuromuscular Disorders 18 (2008) 501–513 2 1. Einleitung 1.1 Die TRP Familie von Kationenkanälen Der erste TRP (Transient Receptor Potential) Kanal wurde in photosensiblen Retinazellen der Fruchtfliege Drosophila melanogaster nachgewiesen. Dieser Kalzium leitende Kanal wird durch Lichteinfall auf den G-Protein-gekoppelten Rhodopsinrezeptor aktiviert (Montell und Rubin 1989). Mittlerweile sind zahlreiche TRP Kanäle bekannt. Ihr Vorkommen ist weit gefächert; es reicht von Hefen und Würmern bis hin zu Wirbeltieren und Säugern. Es handelt sich dabei um Kationen transportierende Kanäle, die hauptsächlich permeabel für Natrium und Kalzium sind. Zu der Superfamilie der TRP Kanäle gehören sieben Unterfamilien, von denen fünf der Gruppe 1 (TRPC, TRPV, TRPM, TRPN und TRPA) und zwei der Gruppe 2 (TRPP und TRPML) zugeordnet werden. Sechs dieser Subfamilien wurden bisher in Säugetieren identifiziert (Montell 2005). TRPN konnte in Würmern, Drosophila und im Zebrafisch nachgewiesen werden (Venkatachalam und Montell 2007). Die phylogenetischen Zusammenhänge sind in Abbildung 1 dargestellt. Abbildung 1.1: Stammbaum der TRP Superfamilie der Säugetiere (Nilius et al. 2007) 3 Die Unterteilung in die zwei Untergruppen basiert auf sequenziellen und strukturellen Unterschieden. Die Gruppe 1 weist eine starke Sequenzhomologie zu den Drosophila TRP Kanälen auf (Montell und Rubin 1989). Allen Kanalproteinen gemein sind sechs Tansmembrandomänen; die Amino- und Carboxyenden liegen bei allen Kanälen intrazellulär. Jeweils vier dieser Untereinheiten lagern sich zu einem funktionellen Kanal zusammen. Der Porus, der die Kationenpassage ermöglicht, liegt zwischen den jeweiligen fünften und sechsten Transmembrandomänen der vier Unterheiheiten (Venkatachalam und Montell 2007). Hinter der sechsten Transmembrandomäne am Carboxyende besitzen die Kanäle TRPC, TRPM und TRPN eine TRP Domäne, die aus einer Folge von 23-25 Aminosäuren besteht. Am N-terminalen Ende der TRPC, TPRV, TRPA und TRPN Subgruppen finden sich Wiederholungen einer Ankyrin Sequenz. (Venkatachalam und Montell 2007) Die Mitglieder der Gruppe 2 haben extrazellulär, zwischen der ersten und zweiten Transmembrandomäne, eine große Schleife (Venkatachalam und Montell 2007) (siehe Abbildung 1.2 a und b). Abbildung 1.2: Aufbau der einzelnen TRP Proteine; (a) Gruppe 1; (b) Gruppe 2; A Ankyrinrepeat; cc coiled- coil Domäne Proteinkinase Domäne (rot); TRP Domäne (blau); P Pore(Venkatachalam and Montell 2007); Cytoplasm= Zytoplasma (Venkatachalam und Montell, 2007). 4 TRP Kanäle kommen in vielen verschiedenen Zell- und Gewebetypen vor. Außerdem sind die Aktivierungsmechanismen sehr variabel und nicht den Untergruppen zuzuordnen. Die Aktivität der TRP Kanäle wird zum einen über Rezeptoren und intrazelluläre Signalkaskaden G-Protein vermittelt über Phosphatidylinositolbisphosphat (PIP2), Diacylglycerol (DAG) und Inositoltrisphosphat (IP3) modifiziert. Zum anderen ist eine Aktivierung durch exogene (z.B. Capsaicin) oder endogene (z.B. Anandamid) Liganden möglich. Einige der Kanäle können auch direkt
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