J. Blanco, "Nuevas técnicas de imagen..."
Anuncio
Nuevas Técnicas de Imagen y su Contribución al Area de la Biomedicina Jerónimo Blanco, Nuria Rubio, Ramón Ayats, Marta Vilalta, Irene Román Juli Rodríguez. Centro de Investigación Cardiovascular CSIC-ICCC Barcelona Nanomedicinas y Medicinas Innovadoras UIMP Santander 14-18 Agosto 2006 Los PROCEDIMIENTOS de IMAGEN NO INVASIVOS permiten VISUALIZAR y CUANTIFICAR la ESTRUCTURA / FUNCIÓN sin necesidad de DESTRUIR al SUJETO EXPERIMENTAL Penetración del Espectro Electromagnético en el Cuerpo Humano Implicaciones para la Generación No-Invasiva de Imagenes MRI RESONANCIA MAGNETICA NUCLEAR IMAGEN FOTONICA CAT PET TOMOGRAFIA POR EMISIÓN DE POSITRONES Principles of Magnetic Resonance PRECESSION FREQUENCY INTENSITY OF THE MAGNETIC FIELD GYROMAGNETIC RATIO = MAGNETIC MOMENT/ANGULAR MOMENT (For 1H (proton) = 63,9 MHz in a 1,5 Tesla field) Chris Boesch, Molecular Aspects of Medicine 20 (1999) 185-318 Generation of MR Images MRI Imaging of Mesenchymal Cells “Passively” Labelled with Ferromagnetic Particles Fast gradient-recalled echo FGRE) delayed-enhancement MRI (DE-MRI) fastspin echo (FSE) 10X107 Feridox-labelled MSCs 24 hours P.I. 1 week P.I. Prussian Blue staining DiI (I) and DAPI Kraitchman, D. et al, Circulation May 13, 2290-2293 ( 2003) Ferritin as an Endogenous Reporter Protein for MRI Cohen B. et al, Neoplasia . Vol. 7, No. 2, February 2005, pp. 109 – 117 MRI Detection of Iron Accumulation Resulting from induction of Ferritin Expression GLYOMA SUBCUTANEOUS TUMORS (MICE) PLUS OR MINUS INDUCTION OF FERRITIN EXPRESSION ADENOVIRUS VECTOR FOR FERRITIN EXPRESSON UNDER CONTROL OF THE CMV PROMOTER DIRECTLY INOCULATED IN A MOUSE BRAIN Cohen B. et al, Neoplasia . Vol. 7, No. 2, February 2005, pp. 109 – 117 Genove, G., Nature Medicine11(4)::450-454 (2005) Principles of Positron Emission Tomography (PET) TO GENERATE AN IMAGE THERE MOST BE A DIFFERENTIAL ACCUMULATION OF ISOTOPE IN THE VOLUME OF INTEREST Half-life of some commonly used positron emitters Radiosotope Half-life 11C 15O 13N 18F 20.3 minutes 2.10 minutes 10.0 minutes 109 minutes Good - Very good penetration required - Tomography - Pharmacy for in situ sinthesis - Hybrid PET/CAT - Possible to analyze gene expression Bad -Exogenous substrate -Very short half-life - Very expensive/test - Expensive instrument Hybrid Functional (18FDG) PET-CAT Imaging allows Anathomic Location of Isotope CAT PET CAT PET/CAT CAT PET PET PET/CAT J. Franquiz et al., Different PET Reporter-Substrate Combinations allow Detection of Gene Expression DUAL REPORTER EXPRESSION STRATEGY 18F-FESP 18F-FHBG DOPAMINE RECEPTOR : 18F-SPIPERONE THYMIDINE KINASE : 18F-GANCICLOVIR Chen I., et al, Circulation March 23,1415-1420 (2004) Reporter Expression Can Be Made Tissue/Organ Specific IN MICE TRANSGENIC FOR LIVER SPECIFIC EXPRESSION OF CRE-LOX RECOMBINASE WHOLE ANIMAL INFECTION WITH ADENOVIRUS CONTAINING “loxP” SEQUENCES RESULTS IN EXCISSION OF INTERFERING loxP FLANKED SEQUENCES OF THE VECTOR AND LIVER SPECIFIC EXPRESSION OF HSVTHYMIDINE KINASE DETECTABLE BY ACCUMULATION OF 18FHBG (GANCICLOVIR) - CRE +VIRUS + CRE +VIRUS + CRE - VIRUS + CRE - VIRUS G. Sundaresan, et al, Gene Therapy 1–10 (2004) Three Different Reporter Gene Vectors Can Be Used to Monitor Different Cell Types in the Same Animal CMV-Red.Fluorescent.Protein CMV-Thymidine.Kinase CMV-Renilla.Luciferase CMV-Renilla.Luciferase-Thymidine.Kinase-Red.Fluorescent.Protein A SINGLE MULTIGENE CONSTRUCT UNDER THE SAME PROMOTER CAN BE USED TO MONITOR A CELL TYPE USING THREE DIFFERENT IMAGING MODALITIES P. Ray, et al, [CANCER RESEARCH 64, 1323–1330, February 15, 2004] Principles of Bioluminescence + ATP + ADP PHOTINUS PYRALIS Eficiencia Cuántica (%) Low Light Level Imaging System HAMAMATSU Photonics Longitud de Onda Experimental Model Expression Vectors Viral Particles Packaging Labelled MSC EGFP: Constitutive expression Transduction reporter Transducción Photinus Pyralis Luciferase: Constitutive expression Proliferation reporter Specific Promoter Renilla Reniformis Luciferase: Tissue specific expression Differentiation reporter Cell implantation Animal Model Non-invasive Imaging System Modelo Experimental Partículas infecciosas Tipos celulares Vectores de expresión Empaquetamiento Transducción Célula precursora marcada Expresión de: EGFP: Expresión constitutiva Monitorizar transducción GFP constitutiva Luciferasa P constitutiva Luciferasa R regulada GFP Promotor específico Luciferasa Renilla Reniformis: Expresión específica de tejido Monitorizar diferenciación Modelo animal R.luc F.luc Luciferasa Photinus Pyralis: Expresión constitutiva Monitorizar proliferación PROCEDIMIENTO NO-INVASIVO DE IMAGEN Infarto experimental Implantación de células Demonstration of Promoter Function CL1 CELLS SPONTANEOUSLY DIFFERENTIATE INTO CHONDROCYTES AND EXPRES COLLAGEN TRANSDUCE CELLS WITH A LENTIVIRAL VECTOR FOR EXPRESSION OF THE LUCIFERASE GENE UNDER CONTROL OF THE PROMOTER FOR COLLAGEN 2A1 SEED ONE CELL/WELL ALLOW SINGLE CELLS TO GROWTH AND FORM COLONIES ANALYZE LIGHT PRODUCTION CAPACITY SOME COLONIES, INITIALLY SILENT, SPONTANEOUSLY DIFFENTIATE AND START PRODUCING LIGHT Relationship between video-response and cell number PHOTON COUNTSS 60000 70 35 17 8 4 0 R 2 = 0,942 50000 40000 30000 20000 10000 0 0 NUMBER OF CELLS 10 20 104 105 106 107 PHOTON COUNTS 103 105 R2 = 0,881 100000 10000 1000 100 106 107 NUMBER OF CELLS 10000000 2 R = 0,8416 1000000 100000 10000 1000 100 10 + RELATIVE INTENSITY 1 1 10 10 1 1 00 000 000 000 000 0 00 0 00 00 00 0 CELL NUMBER 10 PHOTON COUNTS 104 70 1000000 100000000 103 50 60 10 NUMBER OF CELLS 102 40 CELL NUMBER 10000000 102 30 - 10 0 10 1 1 00 1000 000 1000 000 00 00 00 0 0 0 CELL NUMBER 0 A B 4 WEEK-DORSAL C 6 WEEK-DORSAL E D 6 WEEK LUMBAR L N 6 WEEK-VENTRAL F G Noninvasive Imaging 10 WEEK-VENTRAL LUMBAR L.N. LUNGS H I J THORACIC CAVITY THORACIC L. N. 16 WEEK PROSTATE IMPLANTED TUMOR BEFORE CONTROL Differential Effects of Paclitaxel Treatment (10mg/kg/day) on Various Human Prostate Tumors AFTER CONTROL PACLITAXEL PACLITAXEL 40356 326554819 683636869 6746377410 PC 3.Sluc 6353789 9724399919 RELATIVE CHANGE CONTROL PACLITAXEL 350 BEFORE 300 % CHANGE 250 AFTER PC 3.Sluc 50 200 PACLITAXEL CONTROL PACLITAXEL 150 % CHANGE 100 AFTER DU 145.Sluc 50 0 -50 -100 -150 PACLITAXEL 200 150 100 % CHANGE CONTROL BEFORE 0 PACLITAXEL BEFORE CONTROL AFTER 150 100 CONTROL LNCaP.sluc 200 50 0 -50 CONTROL -100 -150 PACLITAXEL Non Invasive Detection of C3H10T1/2 in Mouse Heart •C3H/10T1/2 cels labelled with Retro-EGFP-Fluc vector •Injected in the left ventricle wall (2.105 cells in 3µl volume) •Luciferin tail vein injected •Deteccion in Low Light Level Imaging System HAMAMATSU Photonics Detección de las Células C17-2 Marcadas con el Vector pr-EGFP-CMV-Fluc Inoculadas en Cerebro de Ratón 8x8 1x1 5000 células 10000 células I.P. I.V. Detection of C3H10T1/2 Cells in Muscle. Standard Curve 1000 5000 10000 50000 14000000 y = 23,404x - 35618 R2 = 0,9999 12000000 Foton Counts 500 10000000 8000000 6000000 4000000 2000000 0 0 200000 400000 Inoculated Cells 600000 100000 500000 Estudio de la Capacidad de Proliferación Celular sobre Diferentes Scaffolds (1) Day 0 Day 7 Day 14 Day 30 Day 45 Day 90 Alginato PEG Hydrogel 1 Quitosano No Scaffold Estudio de la Capacidad de Proliferación Celular sobre Diferentes Scaffolds (2) Day 0 Day 7 Day 14 Day 30 Day 45 Microcarriers 1 Microcarriers 2 PEG Hydrogel 2 Quitosano Micropolvo Recovery of Cells from Scaffolds Grown In vivo - Cells recovered from explants are green and produce light from luciferase - Still unresolved: amount of light/cell, due to difficulties growing explanted cells Muscle explant Visible light microscopy Fluorescence microscopy Luciferase luminescence of cultivated cells from explants Autofluorescence of muscle tissue Whole Body Distribution of C3H10T1/2 Cells Accumulation of cells in the lungs 20000000 120000000 Number of Photons Number of Photons 18000000 100000000 16000000 14000000 80000000 12000000 10000000 60000000 8000000 40000000 6000000 4000000 20000000 2000000 660 5 30 d6i 5 as 0 550 5 565 0 40 440 5 455 3 30 0 35 35 220 0 2 25 5 Labeled cells + Luciferin 55 110 0 115 5 00 00 Minutos Post-Inoculation Minutos Post-Inoculation 30 days post-inoculation Lungs Hepatic nodules BALB/c homozygous nude (nu/nu) mice
