Immunomodulatory function of astrocytes [Elektronische Ressource] : characterization and regulation of prostanoid metabolism and cytokine synthesis / vorgelegt von Sonja Johann

Sonja Johann

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2009
• Nombre de lectures: 32
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Immunomodulatory function of astrocytes:
characterization and regulation of
prostanoid metabolism and cytokine synthesis





Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH
Aachen University zur Erlangung des akademischen Grades eines Doktors der
Naturwissenschaften genehmigte Dissertation

vorgelegt von

Diplom-Biologin
Sonja Johann
aus Simmern (Hunsrück)




Berichter: Universitätsprofessor Dr. Herrman Wagner
Universitätsprofessor Dr. Cordian Beyer

Tag der mündlichen Prüfung: 28.05.2009






Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. CONTENTS
ABBREVIATIONS ....................................................................................................................... 1
1. GENERAL INTRODUCTION ................................................................................................ 2
1.1 INFLAMMATION..................................................................................................................... 2
1.2 NEUROINFLAMMATION......................................................................................................... 2
1.3 ASTROCYTES DURING CNS PATHOLOGY ............................................................................. 6
1.4 INFLAMMATORY MEDIATORS IN THE CNS........................................................................... 8
1.5 LIPOPOLYSACCHARIDE (LPS)-INDUCED INFLAMMATION................................................. 11
1.6 NUCLEAR HORMONES 12
1.7 NUCLEAES AS MODULATOR OF NEUROINFLAMMATION................................... 15
2. SCOPE OF THE STUDY....................................................................................................... 18
3. MATERIAL AND METHODS.............................................................................................. 19
3.1 PRIMARY ASTROCYTE CULTURE ........................................................................................ 19
3.2 TREATMENT OF CELL CULTURE ......................................................................................... 19
3.3 AFFYMETRIX GENE CHIP ARRAY ...................................................................................... 20
3.4 QUANTITATIVE RT-PCR.................................................................................................... 20
3.5 SDS- PAGE AND WESTERN BLOT 21
3.6 TNF-α, IL-6 AND PGE IMMUNOASSAY............................................................................ 22 2
3.7 ANTIBODY ARRAY............................................................................................................... 22
3.8 LDH ASSAY ......................................................................................................................... 23
3.9 LIFE DEATH ASSAY.............................................................................................................. 23
4. EXPRESSION OF ENZYMES INVOLVED IN THE PROSTANOID METABOLISM
BY CORTICAL ASTROCYTES AFTER LPS-INDUCED INFLAMMATION.................. 24
4.1 ABSTRACT ........................................................................................................................... 24
4.2 INTRODUCTION ................................................................................................................... 25
4.3 MATERIAL AND METHODS ................................................................................................. 27
4.4 RESULTS .............................................................................................................................. 29
4.5 DISCUSSION ......................................................................................................................... 33
5. ANTI-INFLAMMATORY EFFECT OF RETINOIC ACID ON PROSTAGLANDIN
SYNTHESIS IN CULTURED CORTICAL ASTROCYTES................................................. 37
5.1 ABSTRACT 37
5.2 INTRODUCTION ................................................................................................................... 38 5.3 MATERIAL AND METHODS ................................................................................................. 40
5.4 RESULTS .............................................................................................................................. 42
5.5 DISCUSSION ......................................................................................................................... 52
5.6 ACKNOWLEDGEMENTS ....................................................................................................... 57
6. BRAIN REGION-SPECIFIC ASTROGLIAL RESPONSES IN VITRO AFTER LPS
EXPOSURE ................................................................................................................................. 58
6.1 ABSTRACT ........................................................................................................................... 58
6.2 INTRODUCTION ................................................................................................................... 59
6.3 MATERIAL AND METHODS ................................................................................................. 60
6.4 RESULTS .............................................................................................................................. 62
6.5 DISCUSSION ......................................................................................................................... 68
7. ESTROGEN AND PROGESTERONE REDUCE LPS-INDUCED EXPRESSION OF
TNF-ALPHA AND INTERLEUKIN-18 IN MIDBRAIN ASTROCYTES ........................... 72
7.1 ABSTRACT ........................................................................................................................... 72
7.2 INTRODUCTION ................................................................................................................... 73
7.3 MATERIAL AND METHODS ................................................................................................. 74
7.4 RESULTS .............................................................................................................................. 74
7.5 DISCUSSION ......................................................................................................................... 77
7.6 ACKNOWLEDGMENTS ......................................................................................................... 78
8. GENERAL DISCUSSION...................................................................................................... 79
9. SUMMARY 83
10. ZUSAMMENFASSUNG 85
11. ACKNOWLEDGEMENT.................................................................................................... 87
12. REFERENCES...................................................................................................................... 88






LIST OF FIGURES
Figure 1. The neurovascular unit..................................................................................................................5
Figure 2. Cell-surface recognition of LPS..................................................................................................12
Figure 3. Retinoic acid synthesis and signaling .........................................................................................14
Figure 4. Cellular signaling and transduction mechanisms of steroid hormones .......................................15
Figure 5. The effect of LPS-treatment on the expression pattern of enzymes involved in prostanoid
metabolism ..................................................................................................................................................31
Figure 6. Time-course of LPS-induced (12 h, 100 ng/ml) stimulation of COX-2 (A) and COX-1 (B)
mRNA expression, and production of PGE (C) in cortical astrocytes .......................................................32 2
Figure 7. The effect of dexamethasone (DEX) and the COX-2 inhibitor NS-398 on LPS-mediated
regulation of enzymes involved in prostanoid metabolism as well as on PGE production in cortical 2
astrocytes......................33
Figure 8. Treatment of astrocytes with lipopolysaccharides (LPS) and retinoic acid (RA) does not affect
cell viability..................45
Figure 9. Prostaglandin (PG) synthesis in cortical astrocyte cultures ........................................................45
Figure 10. Retinoic acid (RA) influences the effects of lipopolysaccharide (LPS) on expression of
enzymes necessary for prostaglandin metabolism. .....................................................................................47
Figure 11. Enzyme expression of leukotriene metabolism is reduced by lipopolysaccharide (LPS) but not
affected by additional retinoic acid (RA) treatment. ...................................................................................48
Figure 12. Retinoic acid (RA) reduces the lipopolysaccharide (LPS) - dependent protein expression of
cyclooxygenase-2 (COX-2).........................................................................................................................49
Figure 13. Retinoic acid reduces the LPS-induced synthesis of prostaglandin E2. Astrocyte cultures were
treated as described for gene expression analysis. ..