Decoy gelatin nanoparticles as a novel tool to elucidate the role of {NF-_k63B [NF-kappa-B] in Kupffer cells on hepatic ischemia, reperfusion injury [Elektronische Ressource] / Florian Hoffmann

Florian Hoffmann

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München











Decoy gelatin nanoparticles as a novel tool to elucidate
the role of NF- κB in Kupffer cells on hepatic
ischemia/reperfusion injury



Florian Hoffmann
aus München
2007
Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Frau Prof. Dr. Angelika M. Vollmar betreut.



Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.



München, am 24.04.2007



(Florian Hofman)




Dissertation eingereicht am 04.05.2007
1. Gutachter Frau Prof. Dr. Angelika M. Vollmar
2. Gutachter Herr Prof. Dr. Gerhard Winter
Mündliche Prüfung am 26.06.2007


Contents I
1 CONTENTS
II 1 Contents

1 CONTENTS ........................................................................................................ I
2 INTRODUCTION ..............................................................................................1
2.1 Background and aim of the study .............................................................2
2.2 Kupffer cells ................................................................................................3
2.3 NF- κB...........................................................................................................5
2.3.1 General aspects.....................................................................................5
2.3.2 NF- κB in the liver.................................................................................7
2.3.3 NF- κB inhibitors...................................................................................8
2.4 Decoy oligodeoxynucleotides .....................................................................9
2.5 Carriers .....................................................................................................11
2.5.1 Targeting of Kupffer cells ..................................................................11
2.5.2 Carriers for oligonucleotides..............................................................12
2.5.3 Gelatin nanoparticles..........................................................................13
2.6 LPS.............................................................................................................14
2.7 Hepatic ischemia/reperfusion injury ......................................................15
2.7.1 General mechanisms...........................................................................15
2.7.2 Role of NF- κB ....................................................................................18
2.7.3 Interventions.......................................................................................19
3 MATERIALS AND METHODS.....................................................................21
3.1 Materials and solutions ............................................................................22
3.2 Decoy oligodeoxynucleotides ...................................................................22
3.3 Gelatin nanoparticles ...............................................................................23
3.3.1 Materials.............................................................................................23
3.3.2 Manufacture of decoy nanoparticles...................................................23
3.3.3 Preparation of fluorescent cationic gelatin nanoparticles...................25 Contents III
3.4 DOTAP/DOPC liposomes........................................................................ 25
3.5 Cell culture................................................................................................ 25
3.5.1 Materials.............................................................................................26
3.5.2 Culture of RAW-Macrophages .......................................................... 27
3.5.3 Isolation and culture of Kupffer cells ................................................ 27
3.6 Rat animal models.................................................................................... 28
3.6.1 Materials28
3.6.2 Biodistribution studies.......................................................................28
3.6.3 LPS.....................................................................................................29
3.6.4 Ischemia/reperfusion injury...............................................................29
3.7 Immunohistochemistry............................................................................ 31
3.7.1 Materials.............................................................................................31
3.7.2 Antibodies..........................................................................................31
3.7.3 Staining of isolated Kupffer cells....................................................... 31
3.7.4 Staining of liver tissue........................................................................ 32
3.7.4.1 p65..................................................................................................32
3.7.4.2 Distribution of labeled nanoparticles ............................................. 33
3.7.4.3 Distribution of labeled decoy nanoparticles................................... 33
3.8 EMSA ........................................................................................................ 33
3.8.1 Materials and solutions ...................................................................... 33
3.8.2 Extraction of nuclear protein from RAW 264.7 macrophages .......... 35
3.8.3 Extraction of nuclear protein from liver tissues................................. 35
3.8.4 Protein quantification.........................................................................36
3.8.5 Radioactive labeling of consensus oligonucleotides.......................... 36
3.8.6 Binding reaction and electrophoretic separation................................ 36
3.8.7 Detection and evaluation.................................................................... 37
3.9 Real time RT-PCR ................................................................................... 37
3.9.1 Primers...............................................................................................37
3.9.2 RNA isolation and sample preparation .............................................. 38
3.9.3 Reverse transcription..........................................................................38
3.9.4 Real-time PCR39 IV 1 Contents
3.9.5 Quantification.....................................................................................39
3.10 ELISA ........................................................................................................39
3.11 Measurement of transaminases...............................................................40
3.12 Gene Chip analysis ...................................................................................40
3.12.1 Isolation of RNA................................................................................40
3.12.2 Reverse transcription and hybridization.............................................41
3.13 Statistical analysis.....................................................................................41
4 RESULTS..........................................................................................................43
4.1 In vitro uptake: isolated Kupffer cells....................................................44
4.2 In vivo distribution...................................................................................45
4.2.1 Gelatin nanoparticles..........................................................................45
4.2.2 Decoy ...........................................................................45
4.2.2.1 Systemic distribution......................................................................46
4.2.2.2 Intrahepatic localization.................................................................47
4.2.3 DOTAP/DOPC liposomes..................................................................47
4.3 In vitro: Decoy nanoparticles and LPS – RAW macrophages .............48
4.3.1 Composition of the NF-κB dimer in LPS challenged RAW
macrophages .......................................................................................................48
4.3.2 NF- κB decoy oligonucleotides bind NF- κB.......................................49
4.3.3 Decoy nanoparticles reduce LPS induced NF- κB activity in vitro ....50
4.4 In vivo: Decoy nanoparticles and LPS....................................................51
4.4.1 Induction of NF- κB activity by LPS ..................................................52
4.4.2 Influence on NF-κB activity...............................................................52
4.4.3 Decoy nanoparticles retain p65 within the cytoplasm........................53
4.4.4 Influence on TNF- α ............................................................................54
4.4.4.1 TNF- α mRNA expression...............................................................54
4.4.4.2 TNF- α release .................