Uptake mechanism of Hepatitis B virus into susceptible primary hepatocyte cultures [Elektronische Ressource] = Aufnahmemechanismus des Hepatitis-B-Virus in suszeptible primäre Hepatozyten-Kulturen / vorgelegt von Nicole Kott

Kott, Pierre Nicole

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2010
• Nombre de lectures: 34
• Poids de l'ouvrage: 5 Mo

Extrait

Angefertigt am
Fachbereich 08-Biologie und Chemie
in Zusammenarbeit mit dem
Institut für Medizinische Virologie
der Justus-Liebig-Universität Gießen



Uptake mechanism of Hepatitis B Virus into
susceptible primary hepatocyte cultures

Aufnahmemechanismus des Hepatitis B Virus in
suszeptible primäre Hepatozyten Kulturen



Inauguraldissertation
zur Erlangung des akademischen Grades
d o c t o r r e r u m n a t u r a l i u m
(Dr. rer. nat.)
des Fachbereichs 08-Biologie und Chemie
der Justus-Liebig-Universität Gießen



vorgelegt von
Dipl.-Biol. Nicole Kott



Gießen 2010


















Dekan: Prof. Dr. Volkmar Wolters


Gutachter: Prof. Dr. Michael Martin
Prof. Dr. Dr. h. c. Wolfram Gerlich






Tag der Disputation: 14.01.2011
-Index-
Index

Abbreviations.......................................................................................... II

Summary................................................................................................IV

Zusammenfassung .................................................................................. V

1. Introduction ...................................................................................... 1
1.1 Endocytosis.......................................................................................... 1
1.1.1 Entry of pathogens .................................................................................................2
1.2 Pathways of entry into cells.................................................................. 2
1.2.1 Dynamin-dependent endocytosis .........................................................................3
1.2.1.1 Clathrin-mediated endocytosis......................................................................3
1.2.1.2 Caveolin-mediated uptake .............................................................................3
1.2.1.3 Other pathways...............................................................................................4
1.2.2 Dynamin-independent endocytosis ......................................................................4
1.2.2.1 Phagocytosis....................................................................................................4
1.2.2.2 Macropinocytosis............................................................................................5
1.2.2.3 CLIC-GEEC .....................................................................................................6
1.2.2.4 Other pathways...............................................................................................6
1.2.3 pH dependence of viral infection ..........................................................................7
1.2.4 Role of the cytoskeleton for endocytic pathways .................................................7
1.3 Hepatitis B virus (HBV) .......................................................................8
1.3.1 Taxonomy ...............................................................................................................8
1.3.2 Transmission and disease......................................................................................8
1.3.3 Prevention and therapy .........................................................................................9
1.3.4 Morphology...........................................................................................................10
1.3.5 Genome.................................................................................................................12
1.3.6 Viral life-cycle....................................................................................................... 14
1.3.7 Model systems for HBV infection ....................................................................... 16
1.4 Aim of this work..................................................................................17

2. Material ........................................................................................... 18
2.1 Buffer, solutions and media ............................................................... 18
2.1.1 Buffers...................................................................................................................18
2.1.2 Solutions ...............................................................................................................18
2.1.3 Media.....................................................................................................................19
2.2 Chemicals ..........................................................................................20
2.3 Inhibitors............................................................................................21
2.3.1 Pharmacological inhibitors..................................................................................21 -Index-
2.3.2 myr preS1-HBV peptide.......................................................................................21
2.4 Viruses................................................................................................21
2.4.1 HBV.......................................................................................................................21
2.4.2 HBsAg ...................................................................................................................21
2.4.3 Control viruses ..................................................................................................... 21
2.5 Cell Culture........................................................................................ 22
2.5.1 Cell culture systems .............................................................................................22
2.5.2 Cells.......................................................................................................................22
2.5.2.1 Primary Tupaia hepatocytes (PTH) ............................................................22
2.5.2.2 HepG2 ...........................................................................................................23
2.5.2.3 HepaRG.........................................................................................................23
2.5.2.4 BHK ...............................................................................................................23
2.5.2.5 CV-1 ...............................................................................................................23
2.5.2.6 Huh-7.............................................................................................................23
2.5.2.7 HeLa ..............................................................................................................23
2.6 Antibodies and –sera ......................................................................... 23
2.6.1 Primary antibodies and –sera .............................................................................23
2.6.2 Secondary antibodies...........................................................................................24
2.7 Fluorescent dyes ................................................................................ 24
2.8 DNA-and protein standards ............................................................... 24
2.9 Plasmids ............................................................................................ 24
2.10 Commercial kits................................................................................. 24
2.11 PCR .................................................................................................... 24
2.11.1 X-PCR....................................................................................................................24
2.11.2 cccDNA-PCR.........................................................................................................24
2.11.3 SV40-PCR.............................................................................................................25
2.11.4 Primer and Hybridisation probes (Hybprobes).................................................25
2.11.5 Mastermixes .........................................................................................................25

3. Methods ...........................................................................................26
3.1 Cell culture......................................................................................... 26
3.1.1 Isolation of PTH ...................................................................................................26
3.1.2 Cultivation of stable cell lines..............................................................................27
3.1.2.1 BHK21, CV-1, HeLa, HepG2, Huh-7 ...........................................................27
3.1.2.2 HepaRG.........................................................................................................27
3.2 Virus preparation .............................................................................. 27
3.2.1 HBV.......................................................................................................................27 <