Regulation of suppressor of cytokine signalling 3 expression by the pro-inflammatory cytokine {(IL-1β) [(IL-1beta)] in vitro and inflammatory stimuli in vivo [Elektronische Ressource] / vorgelegt von Xiangping Yang

Labor 15

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Regulation of suppressor of cytokine signalling 3 expression by
the pro-inflammatory cytokine (IL-1 β) in vitro and inflammatory
stimuli in vivo



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften genehmigte Dissertation



vorgelegt von


Master of Medicine
Xiangping Yang
aus Tianmen (Hubei province, VR China)




Berichter: Universitätsprofessor Dr. Peter C. Heinrich
prof. Fritz M. Kreuzaler


Tag der mündlichen Prüfung: 20. Oktober 2005





Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar















Publications from this work

1. Xiang-Ping Yang, Ute Albrecht, Vera Zakowski, Radoslaw M. Sobota,
Dieter Häussinger, Peter C. Heinrich, Stephan Ludwig, Johannes G. Bode, and
Fred Schaper. (2004)
Dual function of interleukin-1beta for the regulation of interleukin-6-induced
suppressor of cytokine signalling 3 expression. J Biol Chem; 279: 45279-89

2. Xiang-Ping Yang, Fred Schaper, Andreas Teubner, Peter C. Heinrich,
Siegfried Matern and Elmar Siewert. (2005)
Interleukin-6 plays a crucial role in the hepatic expression of SOCS3 during
acute inflammatory processes in vivo. J Hepatol; 43: 704-10

Further publications:

3. Yan Su, Jilin Wang, Xiang-Ping Yang, Honggang Xue, Jiahong Zhu. (2001)
Study on purification and immunogenecity of Rabies virus nucleoprotein.
Virologica Sinica; 16: 64-67

Table of Content

Abbreviations: ...................................................................................V
1 Introduction ...............................................................................1
1.1 Acute phase response .............................................................................. 1
1.2 IL-1 signalling pathway .......................................................................... 2
1.2.1 IL-1 family cytokines .............................................................................................. 2
1.2.2 signalling cascades initiated by IL-1....................................................................... 3
1.3 IL-6 signalling pathway 4
1.3.1 IL-6 and IL-6-type cytokines .................................................................................. 4
1.3.2 JAK-STAT pathway................................................................................................6
1.4 Regulators of the JAK-STAT signalling pathway.................................. 8
1.4.1 SHP2........................................................................................................................ 8
1.4.2 PIAS. 10
1.4.3 Suppressors of cytokine signalling (SOCS) .......................................................... 10
1.4.4 Pro-inflammatory cytokines..................................................................................13
1.5 Aims of this study ................................................................................. 14
1.5.1 Regulation of IL-6-induced SOCS3 expression by IL-1 β..................................... 14
1.5.2 Importance of IL-6 on the SOCS3 expression in vivo........................................... 14
2 Materials and Methods ...........................................................16
2.1 Chemicals.............................................................................................. 16
2.2 Radiochemicals ..................................................................................... 16
2.3 Oligonucleotides ................................................................................... 16
2.4 Antibodies and cytokines...................................................................... 16
2.5 Reagents for cell culture ....................................................................... 17
2.5.1 Eukaryotic cells and the culture ............................................................................ 17
2.5.2 Prokaryotic cells and the culture ........................................................................... 18
2.5.3 Preparation of calcium competent E. coli cells ..................................................... 18
2.6 Plasmids ................................................................................................ 19
2.7 Methods of molecular biology .............................................................. 20
2.7.1 Plasmid transformation into CaCl competent E. coli cells .................................. 20 2
2.7.2 Restriction digestion of plasmid DNA .................................................................. 20
ITable of Content

2.7.3 Agarose gel electrophoresis...................................................................................20
2.7.4 DNA purification from agarose gels ..................................................................... 21
2.7.5 DNA ligation.........................................................................................................21
2.7.6 Plasmid mini-preparation and maxi-preparation................................................... 22
2.7.7 Quantification of nucleic acids.............................................................................. 22
2.7.8 Phenol-chloroform-isoamyl alcohol extraction of DNA....................................... 22
2.7.9 Ethanol precipitation of DNA ............................................................................... 22
2.7.10 DNA sequencing...................................................................................................23
2.8 Nuclear extraction and electrophoretic mobility shift assay (EMSA).. 23
2.8.1 Nuclear extraction.................................................................................................23
2.8.2 Oligonucleotides hybridization and labeling......................................................... 24
2.8.3 Electrophoretic mobility shift assay (EMSA) ....................................................... 24
2.9 Reporter gene assay .............................................................................. 25
2.9.1 Transient transfection............................................................................................26
2.9.2 Luciferase-assay....................................................................................................26
2.9.3 β-Galactosidase-assay ........................................................................................... 26
2.10 Preparation of cell lysates for immunoprecipitation............................. 27
2.11 Determination of protein concentration according to Bradford ........... 27
2.12 Biotinylation of antibodies.................................................................... 27
2.13 Immunoprecipitations ........................................................................... 28
2.14 SDS-polyacrylamide gel electrophoresis.............................................. 28
2.15 Semi-dry electrophoretic transfer ......................................................... 29
2.16 Immunblotting....................................................................................... 30
2.17 Chromatin immunoprecipitation (ChIP)............................................... 30
2.17.1 Shearing of genomic DNA by sonication.............................................................. 30
2.17.2 Immunoprecipitation.............................................................................................31
2.17.3 Reverse cross link and DNA extraction ................................................................ 31
2.17.4 PCR and visualization of the bands in the gel....................................................... 31
2.18 mRNA extractions................................................................................. 32
2.19 Northern blotting................................................................................... 32
2.20 cDNA synthesis..................................................................................... 33
2.21 Quantification-PCR (Taqman PCR) ..................................................... 33
IITable of Content

2.21.1 Taqman PCR.........................................................................................................33
2.21.2 Quantification of target gene expression............................................................... 33
3 Results.......................................................................................34
3.1 Dual function of IL-1 β on IL-6-induced SOCS3 expression ............... 34
3.1.1 IL-1 β counteracts IL-6-dependent SOCS3-promoter activation........................... 34
3.1.2 Stable expression of a non-degradable I κB(S/A) mutant prevents IL-1 β to
inhibit IL-6-induced SOCS3 promoter activity..................................................... 35
3.1.3 IL-1 β inhibits IL-6-induced STAT3 phosphorylation and DNA binding
activity independent on NF- κB activation ............................................................ 37
3.1.4 The inhibitory activity of IL-1 β on SOCS3-promoter activation is
time-depende