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Publié par | gottfried_wilhelm_leibniz_universitat_hannover |
Publié le | 01 janvier 2009 |
Nombre de lectures | 11 |
Langue | English |
Poids de l'ouvrage | 9 Mo |
Extrait
Adenovirus and VSV:
investigations on virus-host-interactions
to improve safety and efficacy of oncolytic
viruses
Von der
Naturwissenschaftlichen Fakultät der
Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades
Doktor der Naturwissenschaften
Dr. rer. nat.
genehmigte Dissertation
von
Diplom-Biochemiker Peter Schache
geboren am 02. Januar 1978 in Jena
März 2009
Referent: Prof. Dr. Walter Müller, Medizinische Hochschule Hannover
Koreferent: Prof. Dr. Bernd Otto, Tierärztliche Hochschule Hannover
Tag der Promotion: 02. März 2009
TABLE OF CONTENTS i
Table of contents
1. Abstract.................................................................................................... 1
2. Zusammenfassung ................................................................................... 2
3. Introduction ............................................................................................. 3
3.1 Cancer and tumor development.............................................................................. 3
3.1.1 Cancer............................................................................................................. 3
3.1.2 Model of tumor development......................................................................... 4
3.1.3 Therapeutic treatment strategies..................................................................... 6
3.2 Virotherapy............................................................................................................... 7
3.2.1 Development of cancer therapies by viral means........................................... 7
3.2.2 Strategies for the exploitation of viruses as oncolytic agents ........................ 8
3.3 Adenoviruses as oncolytic agents ............................................................................ 9
3.3.1 The human Adenoviruses type 5.................................................................. 10
3.3.2 Adenoviruses as oncolytic vector................................................................. 10
3.3.3 p53-dependent adenoviral vectors................................................................ 11
3.3.4 The endonucleolytic enzyme I-Sce I ............................................................ 13
3.4 Vesicular Stomatitis Virus (VSV) ......................................................................... 14
3.4.1 Structure of Vesicular Stomatitis Virus........................................................ 14
3.4.1.1 Replication cycle of VSV............................................................................. 14
3.4.1.2 Virus-host-interactions ................................................................................. 15
3.4.1.2.1 VSV replication is highly susceptible to the actions of type I
interferons............................................................................................... 15
3.4.1.2.2 VSV usurps the cellular protein biosynthesis machinery....................... 17
3.4.1.2.3 Induction of apoptosis in VSV-infected cells ........................................ 18
3.4.2 VSV as oncolytic vector............................................................................... 19
4. Objectives.............................................................................................. 21
5. Materials and methods........................................................................... 22
5.1 Materials ................................................................................................................. 22
5.1.1 Cell lines....................................................................................................... 22
5.1.1.1 Purchased/provided cell lines....................................................................... 22
5.1.1.2 Stably transfected cell lines.......................................................................... 22
5.1.2 Bacteria......................................................................................................... 23
5.1.3 Mice.............................................................................................................. 23
5.1.4 Plasmids ....................................................................................................... 23
5.1.4.1 Provided plasmids ........................................................................................ 23
5.1.4.2 Constructed plasmids ................................................................................... 25
5.1.5 Adenoviruses................................................................................................ 28
5.1.5.1 Provided Adenoviral vectors........................................................................ 28
5.1.5.2 Constructed Adenoviral vectors ................................................................... 28
5.1.6 VSV.............................................................................................................. 29
5.1.7 Oligonucleotides........................................................................................... 29
5.1.8 Antibodies .................................................................................................... 31
5.1.8.1 Primary antibodies........................................................................................ 31
5.1.8.2 Secondary antibodies.................................................................................... 31
5.1.9 Chemicals ..................................................................................................... 32
5.1.10 Molecular weight standards ......................................................................... 32
5.1.11 Enzymes ....................................................................................................... 32
5.1.12 Kits ............................................................................................................... 33
TABLE OF CONTENTS ii
5.1.13 Devices ......................................................................................................... 33
5.1.14 Media and buffers......................................................................................... 33
5.2 Cell biological methods .......................................................................................... 36
5.2.1 Cell culture techniques ................................................................................. 36
5.2.2 Transfection of cell lines .............................................................................. 36
5.2.2.1 Lipofectamin2000 ........................................................................................ 36
5.2.2.2 Calciumphosphate ........................................................................................ 36
5.2.2.3 Polyethylenimine (PEI) ................................................................................ 36
5.2.3 Microscopical methods ................................................................................ 37
5.2.3.1 Fluorescence microscopy ............................................................................. 37
5.2.3.2 Confocal Laser Scanning Microscope (CLSM) ........................................... 37
5.2.4 Tissue staining.............................................................................................. 37
5.2.4.1 Haematoxylin/Eosin (HE) ............................................................................ 38
5.2.4.2 Immune histochemistry................................................................................ 38
5.2.4.3 TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)
staining ......................................................................................................... 38
5.3 Protein biochemical methods ................................................................................ 38
5.3.1 Preparation of protein extracts from cell culture.......................................... 38
5.3.2 Determination of protein concentration ....................................................... 39
5.3.3 SDS-PAGE and western blot analysis ......................................................... 39
5.3.4 Luciferase assays.......................................................................................... 39
5.3.4.1 Firefly........................................................................................................... 39
5.3.4.2 Dual luciferase reporter system.................................................................... 40
5.3.5 β-Galactosidase assay................................................................................... 40
5.3.6 Caspase-3-activation assay........................................................................... 40
5.4 Molecular biological methods ............................................................................... 41
5.4.1 DNA amplification and purification ............................................................ 41
5.4.1.1 Mini format .................................................................................................. 41
5.4.1.2 Midi/Maxi format............