DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Svenja Sethmann :
Expression and regulation of the growth and differentiation factor GDF-3
Die Expression und Regulation des Wachstumsfaktors GDF-3

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Abstract

The growth and differentiation factor 3 (GDF-3) is unique among the members of the TGF-super family

The growth and differentiation factor 3 (GDF-3) is unique among the members of the TGF- super family. It is almost exclusively expressed in lymphoid tissues (Mc Pherron and Lee, 1993), suggesting a role for GDF-3 in the maintenance and/or regulation in the immune system.

 

Combining MACS technology, quantitative RT-PCR, and in situ hybridisation, the cellular source of GDF-3 secretion within primary and secondary lymphoid tissues could be identified. Magnetic cell sorting (MACS) was applied to separate distinct cell populations according to their specific surface markers to a high purity. mRNA was isolated from the different cell types, reversely transcribed into cDNA, and analysed by quantitative, sequence-specific PCR utilising the LightCycler system. mRNA from total lymphoid tissues was isolated for comparison. Prior to quantification, all samples were normalised to expression of the housekeeping gene ?-Actin. GDF-3 transcript could specifically be detected in B220/Thy1.2/CD11b/CD11c/CD45 negative, adherently growing cells. In situ hybridisation of spleen sections revealed the morphological pattern of the GDF-3 expressing cells. They were clearly identified as stromal cells of the red pulp.

 

To address the question whether GDF-3 expression is regulated, a series of in vitro experiments was performed. Stimulation of splenocytes with Lipopolysaccharide (LPS), a bacterial antigen that activates the immune response, leads to a dramatic downregulation of GDF-3 expression. Furthermore, CD11b+ cells, CD11c+ cells, and DX5+ cells were found to be required to maintain GDF-3 expression in culture. Immunohistochemistry showed that CD11b+ cells are co-localised with the GDF-3 expressing stromal cells in the red pulp of spleen, suggesting an immediate effect for CD11b+ cells in the regulation of GDF-3 expression. If added to CD11b-depleted splenocytes, the CD11b+ fraction, but not conditioned medium from total spleen can restore full GDF-3 expression. Therefore, GDF-3 expression is mediated by direct cell contact rather than by a secreted factor.

 

In conclusion, the data provides strong evidence that GDF-3 is part of the complex ensemble of secreted proteins that govern the immune function.

 

Table of Contents

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0. Titelblatt

1. Einleitung

1.1 Das Immunsystem

1.1.1 Allgemeiner Aufbau und Funktion des Immunsystems

1.1.2 Aufbau und Funktion der Milz

1.2 Die TGF-beta Superfamilie

1.3 Der Wachstums- und Differenzierungsfaktor GDF-3

1.4 Ziel der Arbeit

2. Material und Methoden

2.1 Material

2.1.1 Murines und humanes Gewebe

2.1.2 Antikörper und MACS MicroBeads

2.1.3 Plasmide

2.1.4 Oligonukleotide

2.1.5 Geräte

2.1.6 Enzyme

2.1.7 Reaktionskits

2.1.8 Puffer

2.1.9 Chemikalien und Radiochemikalien

2.1.10 Medien

2.1.11 Kunststoffartikel und sonstige Verbrauchmaterialien

2.2 Methoden

2.2.1 Strategie zur Identifizierung der GDF-3 exprimierenden Zellen

2.2.2 Isolation der lymphatischen Gewebe

2.2.3 mRNA Präparation

2.2.4 cDNA Präparation

2.2.5 Zellsortierung (MACS)

2.2.6 Trennung lymphatischer Zellen durch Adhärenz

2.2.7 Durchflusscytometrie (FACS)

2.2.8 Quantitative LightCycler PCR

2.2.9 Anfertigung histologischer Gewebeschnitte

2.2.10 In situ Hybridisierung

2.2.11 Zellkultur und Zellzahlbestimmung

2.2.12 In vitro Stimulation

2.2.13 In vitro Depletionen

2.2.14 Immunohistochemie

2.2.15 Mechanismus der Regulation der GDF-3 Expression

2.2.16 Das GDF-3 'Standard'-Plasmid

3. Ergebnisse

3.1 Etablierung der LightCycler PCR

3.2 GDF-3 Expression in Gesamtgeweben

3.2.1 GDF-3 Expression in Gesamtgeweben der Maus

3.2.2 GDF-3 Expression in Gesamtgeweben des Menschen

3.3 Identifizierung der GDF-3 exprimierenden Zellen

3.3.1 GDF-3 Expression in definierten Zellpopulationen der Milz

3.3.2 GDF-3 Expression in definierten Zellpopulationen des Knochenmarks

3.3.3 Lokalisation der GDF-3 mRNA in histologischen Schnitten der Milz

3.4 Regulation der GDF-3 Expression

3.4.1 In vitro Stimulationen

3.4.2 In vitro Depletionen

3.4.3 Immunohistochemischer Nachweis CD11b positiver Zellen

3.4.4 Regulationsmechanismus der GDF-3 Expression durch CD11b positive Zellen

3.5 GDF-3 Expression in Rag-/- Mäusen

4. Diskussion

4.1 Etablierung der LightCycler PCR

4.2 GDF-3 Expression in Gesamtgeweben

4.3 Identifizierung der GDF-3 exprimierenden Zellen

4.4 Charakterisierung von Regulationsmechanismen der GDF-3 Expression

4.5 GDF-3 Expression in Rag-2-/- Mäusen

4.6 Schlussfolgerung und Ausblick

4.6.1 Schlussfolgerung

4.6.2 Ausblick

5. Zusammenfassung

6. Referenzen

7. Abkürzungen

 
 

 

More Information:

Online available: http://www.diss.fu-berlin.de/2002/90/indexe.html
Language of PhDThesis: german
Keywords: TGF-beta, GDF-3, growth factor, microenvironment, LightCycler
DNB-Sachgruppe: 32 Biologie
Date of disputation: 16-May-2002
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Roland Lauster
Second Referee: Prof. Dr. Volker A. Erdmann
Contact (Author): sethmann@drfz.de
Contact (Advisor): lauster@drfz.de
Date created:29-May-2002
Date available:04-Jun-2002

 

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