Methylphenidat-induzierte Genexpression in ...· Aus der Klinik und Poliklinik für Psychiatrie, Psychosomatik

  • View
    212

  • Download
    0

Embed Size (px)

Text of Methylphenidat-induzierte Genexpression in ...· Aus der Klinik und Poliklinik für Psychiatrie,...

  • Aus der Klinik und Poliklinik fr Psychiatrie, Psychosomatik und Psychotherapie der

    Universitt Wrzburg

    Direktor: Professor Dr. med. Jrgen Deckert

    Methylphenidat-induzierte Genexpression

    in lymphoblastoiden Zellen von adulten ADHS Patienten

    Inaugural-Dissertation

    zur Erlangung der Doktorwrde

    der Medizinischen Fakultt

    der Julius-Maximilians-Universitt Wrzburg

    Vorgelegt von

    Ricarda Schwarz

    aus Hildesheim

    Wrzburg, Januar 2016

  • Referent: Prof. Dr. med. A. Reif

    Korreferent:

    Prof. Dr. med. M. Romanos

    Dekan: Prof. Dr. M. Frosch

    Tag der mndlichen Prfung:

    22.09.2016

    Die Promovendin ist rztin

  • Abkrzungen

    ABI Ankle Brachial Index ACTH Adrenocorticotropes Hormon AD Alzheimer Disease ADHS Aufmerksamkeitsdefizit-/Hyperaktivittssyndrom AGS Activator of G-protein signaling ALAS Aminolvulinat, delta-, synthase 1 ANOVA Analysis of variance APP Amyloid Precurser Protein ASD Autism Spectrum Disorder ATF Aktivator Protein Komponente ATXN Ataxin B2M beta-2-microglobulin BALF EBV-encoded glycoprotein gp 110 Bcl-2 B-cell-lymphoma 2 bHLH basic Helix loop helix BMI Body-mass-Index Bp Basenpaare C(t) treshold cycle (Schwelle) Ca Calcium CA3 Cornu ammonis CAARS Conners Adult ADHD Rating Scales CAG Cytidin-Adenin-Guanin cAMP zyklisches Adenosinmonophosphat CBV Cerebellre Vermis CCD charged coupled device= ladungsgekoppeltes Bauteil CCT Chaperonin-containing-T-complex Polypeptid CD cluster of differentiation Cdk cyclin dependent kinase cDNA complementary DNA cGK zyklische Guanylatcyklase cGMP zyklisches Guanosinmonophosphat CH Calponin hnliche Domne CHOP Chemotherapieschema COOH Carboxylgruppe CREB cAMP-responsive element binding protein CRF Corticoliberin CT treshold cycle (Schwelle) C-Terminus Carboxy-Ende CTRL Control CUMS chronic unpredictable mild stress D2, D4, DRD Dopaminrezeptoren 2, 4

  • DA Dopamin DNA Desoxyribonukleinsure DAT Dopamintransporter dATP desoxyAdenosintrisphosphat DBH Dopamin Beta Hydroxylase dCTP desoxyCytidintrisphosphat DEPC Diethylpyrocarbonat dGTP desoxyGuanosintrisphosphat DMSO Dimethylsulfoxid DNS/ DNA Desoxyribonukleinsure dNTP Desoxyribonukleosidtriphosphate dsDNA doppelstrngige DNA

    DSM-TR Diagnostic and Statistical Manual of Mental Disor-ders - Text revision

    dT desoxy Thymidin dTTP desoxyThymidintrisphosphat E2F eucaryotic transcription factor 2 EBNA EBV-Nuclear-Antigen EBV Epstein-Barr-Virus EDTA Ethylendiamintetraessigsure EEG Elektroenzephalogramm ELK eag like channel EndMT endothelial-to-mesenchymal-transition eNOS endotheliale NOS ERK extracellular regulated MAP kinase EtBr Ethidiumbromid EVA Ethylenvinylacetat E Extinktionskoeffizient F Female FADD Fas-associated death domain protein FBS fetales Rinderkalbsserum (fetal bovine serum) FDR False discovery rate G1 Lcke, gap GABA Gamma-Amino-Buttersure gDNA genomische DNA Geb Geburtsdatum GH Growth Hormon GLUT3 Glukosetransporter 3 GSC Glasgow Coma Scale GS-well gel-stain GTP Guanosintriphosphat GUCY1B3 guanylate cyclase 1, soluble, beta 3 GUSB glucuronidase beta G-well Gel HBA1c glykosyliertes Hmoglobin

  • HDAC Histondeacetylase HEY1 hairy/enhancer-of-split related with YRPW motif 1 hOGG1 8-hydroxyguanin glycosylase 1 HPRT hypoxanthine phosphoribosyltransferase Hsp Hitzeschockprotein HTR1B Serotonin 1B-Rezeptor HTT Serotoninrezeptor HWZ Halbwertszeit ICD International Classification of Diseases IL Interleukin iNOS intrazellulre NOS IQ Intelligenzquotient IRC Interruncalibrator IRS Insulinrezeptorsubstrat iTaq Thermus aquaticus JNK c-Jun N-terminale Kinase, Januskinase Kb kilo Basen kDa kilo Dalton LANP leucine-rich acidic nuclear protein L-Arg L-Arginin LGN Leu-Gly-Asn= Leucin-Glycin-Aparagin LIMMA Linear Models Microarray

    LMP latentes Membranprotein (Latency Membran Pro-tein)

    M Male MAP3K8 mitogen-activated protein kinase kinase kinase 8 Max MYC associated factor X MEF makrolid efflux protein MEK MAP-Kinase Erk Kinase Meth Methamphetamine MiniDips Diagnostische Interview bei psychischen Strungen miRNA micro RNA mM Mikromolar MMLV Moloney Murine Leukemia Virus MPH Methylphenidat mRNA messenger RNA MSY1 Mykoplasma Synoviae NAV neuron navigator nf Sp1/Sp3 nuclear factor speccificity protein

    NFB nuclear factor kappa-light-chain-enhancer of activat-ed B cells

    NGFI-B nerve growth factor induced protein B NICD intrazellulr konstitutiv aktivierte Notch Domne NK Natliche Killerzellen NMDA N-methyl-D-Aspartat

  • nNOS neuronale NOS NO Stickstoffmonoxid NOS NO-Synthetase NRC no reverse control nRT nicht Reverse Trankriptase nTC no template control N-Terminus Stickstoff-Ende OD optische Dichte ODQ Oxadiazoloquinoxalin PBMC peripheral blood mononuclear cells

    PBS Phospaht gepufferte Salzlsung (phosphate buffered saline)

    PCR polymerase chain reaction

    pCREB phosphoryliertes cAMP-responsive element binding protein

    PDE Phosphodiesterase PFC Prfrontaler Cortex PI Phosphatidylinositol PKA Proteinkinase A POMC Proopiomelanocortin Ppi Pyrophosphat PPI Prepulse Inhibition pRB hypophosphoryliertes Retinoblastomprotein QC Quebec qPCR quantitative PCR RA retinoic acid RAINB1 retinoid-acid-induced in neuroblastoma 1 RBPJ recombination signal sequence-binding protein RFU Relative fluorescence unit RIG retinoic acid inducable gene RIN RNA integrity number RMA Robust Multi Array Average RNA Ribonucelic acid ROR related orphan receptor Rpm rounds per minute RPMI Roswell Park Memorial Institute RQI RNA Quality Index rRNA ribosomale RNA RT Reverse Transkriptase RT-PCR Reverse Transkriptase PCR S Svedberg Sedimentationskonstante S Standardabweichung Sap sporulation associated protein SCA1 spinocerebellre Ataxie Gen 1 SDHA succinate dehydrogenase complex, subunit A

  • SEM standard error of the mean sGC lsliche Guanylatcyclase SH small hydrophobic Sigma Signifikanz siRNA small interfering RNA SK Probenabkrzung von Sarah Kittel SLC2A3 Solute carrier SMRTER Retinoid- und Tyroidrezeptoren SNP small nuclear polymorphism SNAP25 Synapsen assoziierter Protein 25 SPSS Statistical Package for the Social Sciences Std. Dev. Standard deviation TAE Tris Acetat EDTA Puffer TAQ Thermus aquaticus TATA-Box DANN-Region mit Thymin-Adenin TBP TATA box binding protein TFRC transferrin receptor TNF Tumor necrosis factor TRADD TNFR-associated death domain protein TRAF TNF-receptor-associated factor Tris Tris-(hydroxymethyl-) aminomethan Tx Zeitpunkt x U Units U Unknown Unc uncortin precurser UTR untranslated region UV ultraviolettes Licht UV/Vis ultra violette/ visible (light) VNTR variable number of tandem repeats VSN Variance stabilizing normalization w/v Massenprozent (weight)/ Volumenprozent (volume) WPW Wolff-Parkinson-White-Syndrom WURS Wender Utah Rating Scale

    YWHAZ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta

  • Inhaltsverzeichnis

    1 Einleitung ....................................................................................................................... 1

    1.1 Aufmerksamkeitsdefizit-/Hyperaktivittssyndrom ................................................. 1

    1.1.1 Geschichte und Epidemiologie ........................................................................ 2

    1.1.2 Klassifikation .................................................................................................... 3

    1.1.1 Molekularbiologie und Pharmakotherapie ..................................................... 5

    1.2 Methylphenidat ...................................................................................................... 5

    1.3 Fragestellung ........................................................................................................... 6

    2 Material und Methoden ................................................................................................ 8

    2.1 Material .................................................................................................................. 8

    2.1.1 Zelllinien ........................................................................................................... 8

    2.1.2 Synthetische Oligonukleotide ........................................................................ 11

    2.1.3 Reaktionskits .................................................................................................. 11

    2.1.4 Chemikalien ................................................................................................... 12

    2.1.5 Spezielle Gebrauchs- und Verbrauchsmaterialien ......................................... 13

    2.1.6 Gerte ............................................................................................................ 14

    2.1.7 Software ......................................................................................................... 15

    2.2 Methoden ............................................................................................................. 16

    2.2.1 Isolierung humaner Lymphozyten ............................................................. 16

    2.2.2 Transformation mit Epstein-Barr-Virus ...................................................... 18

    2.2.3 Herstellung des Kulturmediums ................................................................ 21

    2.2.4 Kultivierung ................................................................................................ 21

    2.2.5 Splitten ....................................................................................................... 22

    2.2.6 Kontamination ........................................................................................... 23

    2.2.7 Kryokonservierung ..................................................................................