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Informations
Publié par | ruprecht-karls-universitat_heidelberg |
Publié le | 01 janvier 2005 |
Nombre de lectures | 10 |
Langue | English |
Poids de l'ouvrage | 2 Mo |
Extrait
Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
presented by
Master of Science in Biotechnology Marta Enderlin
born in G łogów, Poland
Oral examination:
Evaluation of IP-10 and TNFα-transducing parvoviral vectors as
antitumoral agents in animal glioblastoma models
Referees: Prof. Dr. Werner Buselmaier
Prof. Dr. Lutz Gissmann
To my husband Bernhard
TABLE OF CONTENTS I
Table of contents
Summary……………………………………………………………………………………V
Zusammenfassung………….…………………………………………………….………VI
1 Introduction...............................................................................................................1
1.1 Gliomas .............................................................................................................1
1.1.1 Molecular mechanisms involved in glioma development............................2
1.1.2 Glioma-directed therapy (standard therapies)............................................2
1.2 Gene Therapy ...................................................................................................4
1.2.1 Gene transfer-based immunotherapy.........................................................5
1.2.2 Enzyme/prodrug therapy............................................................................6
1.2.3 Transfer of the therapeutic transgenes into cells........................................6
1.2.4 Antisense strategies...................................................................................7
1.2.5 Viral vectors ...............................................................................................7
1.3 Parvoviruses ...................................................................................................13
1.3.1 Structure and properties of the parvoviral particle....................................15
1.3.2 Gene expression......................................................................................15
1.3.3 Parvoviral proteins ...................................................................................16
1.3.4 Oncosuppressive effects of parvoviruses.................................................18
1.3.5 Vectors derived from autonomous parvoviruses ......................................19
1.3.6 Autonomous parvovirus-mediated tumor therapy.....................................20
1.4 Angiogenesis...................................................................................................21
1.4.1 IP-10 as an antiangiogenic factor.............................................................23
1.4.2 Cytokines and antitumor immunity ...........................................................25
1.4.3 TNFα (tumor necrosis factor α)................................................................27
1.5 Aims of work....................................................................................................30
2 Materials and Methods ...........................................................................................31
2.1 Microbiological Methods..................................................................................31
2.1.1 Culture and Cryopreservation of Escherichia coli.....................................31
2.1.2 Culturing of Electro Competent Bacteria ..................................................31
2.1.3 Transformation of E.coli with Plasmid DNA – Electroporation..................32
2.2 Molecular Biology Methods .............................................................................32
2.2.1 DNA Purification, Analysis and Modification ............................................32
2.2.2 RNA Isolation and Analysis......................................................................35
2.3 Biochemical Methods ......................................................................................36 TABLE OF CONTENTS II
2.3.1 Protein Isolation from Mammalian Cells...................................................36
2.3.2 Protein Determination by the Bradford Assay ..........................................37
2.3.3 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) ..........................37
2.3.4 Western Blot Analysis ..............................................................................38
2.4 Cell Culture Methods.......................................................................................39
2.4.1 Maintenance of Cell Lines........................................................................39
2.4.2 Freezing and Thawing Mammalian Cells .................................................40
2.4.3 Methods of Assessing Cell Proliferation and Viability...............................40
2.5 Cell Biology Methods.......................................................................................42
2.5.1 FACS (Fluorescence Activated Cell Sorting)............................................42
2.5.2 Cytotoxicity Assay....................................................................................43
2.5.3 In Vitro Generation of Mouse Dendritic Cells from Bone Marrow.............43
2.5.4 Measurement of FITC-Dextran Uptake ....................................................44
2.6 Virological Methods.........................................................................................44
2.6.1 Production of Recombinant Viruses.........................................................44
2.6.2 Production of Wild Type Viruses ..............................................................45
2.6.3 Purification and Concentration of Virus Stocks ........................................45
2.6.4 Hemaglutination Assay ............................................................................47
2.6.5 Virus Titration Methods47
2.7 Immunological Methods ..................................................................................50
2.7.1 ELISA (Enzyme-Linked Immunosorbent Assay) ......................................50
2.7.2 ELISPOT Technique ................................................................................51
2.7.3 Immunohistochemistry .............................................................................52
2.8 MRI Analysis ...................................................................................................53
2.8.1 Measurement technique...........................................................................53
2.8.2 Data Analysis ...........................................................................................54
2.9 Animal Techniques..........................................................................................55
2.9.1 Experimental Animals ..............................................................................55
2.10 Statistical Methods56
3 List of Abbreviations ...............................................................................................57
4 Results ...................................................................................................................60
4.1 Characterization of parvoviral infection in glioblastoma cells ..........................60
4.1.1 Sensitivity of glioma cells to the infection with wild type parvoviruses......60
4.1.2 Production of progeny wild type viruses by infected glioblastoma cells ...62 TABLE OF CONTENTS III
4.1.3 Infectability of glioblastoma cells..............................................................63
4.2 Cloning of the transgenes into the parvoviral vectors......................................65
4.2.1 Basic vectors............................................................................................65
4.2.2 Cloning of human IP-10 ...........................................................................66
4.2.3 Cloning of mouse TNFα66
4.2.4 RCV contamination ..................................................................................69
4.3 Major viral protein NS1 expression in glioblastoma cells.................................70
4.3.1 Major viral protein NS1 expression ..........................................................70
4.4 Transgene expression in glioblastoma cells....................................................72
4.4.1 pression evaluated by ELISA .............................................72
4.5 Sensitivity of the glioblastoma cells to TNFα...................................................80
4.5.1 Cytotoxicity tests ......................................................................................80
4.5.2 Endogenous expression of TNFα by glioblastoma cells...........................84
4.6 Animal experiments.........................................................................................87
4.6.1 Analysis of growth of human cells in nude mice.......................................87
4.6.2 Antitumor effect of recombinant parvoviruses on human U87 glioblastoma
cells implanted subcutaneously in nude mice.............................................