Molecular and functional analysis of the cross-talk between human preadipocytes, adipocytes and endothelial cells in vitro [Elektronische Ressource] / Isabelle Mack

TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Ernährungsmedizin




Molecular and functional analysis of the cross-talk
between human preadipocytes, adipocytes and
endothelial cells in vitro




Isabelle Mack


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. M. Klingenspor
Prüfer der Dissertation:
1. Univ.-Prof. Dr. J. J. Hauner
2. Univ.-Prof. Dr. H. Daniel


Die Dissertation wurde am 02.08.2010 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 03.01.2011 angenommen.
Table of contents

1 INTRODUCTION..............................................................................1

1.1 Obesity .........................................................................................................1
1.1.1 Prevalence of obesity.......................................................................................2
1.1.2 Obesity-associated diseases .............................................................................2
1.1.3 Obesity as an inflammatory state .....................................................................4

1.2 Adipose Tissue .............................................................................................5
1.2.1 Adipose tissue function ...................................................................................5
1.2.2 WAT secretory function ..................................................................................6
1.2.2.1 Leptin..........................................................................................................7
1.2.2.2 Adiponectin.................................................................................................7
1.2.2.3 IL-6 .............................................................................................................8
1.2.2.4 Vascular endothelial growth factor (VEGF).................................................8
1.2.2.5 Chemokines.................................................................................................9
1.2.3 WAT morphology ...........................................................................................9
1.2.4 WAT growth and vasculature ........................................................................10
1.2.4.1 Blood vessels, endothelial cells and endothelial dysfunction......................10
1.2.4.2 WAT growth .............................................................................................11
1.2.4.3 Adipogenesis .............................................................................................11
1.2.4.4 WAT vasculature.......................................................................................12
1.2.5 WAT blood flow ...........................................................................................13
1.2.6 WAT hypoxia................................................................................................14
1.2.7 WAT immune cell infiltration........................................................................16
1.2.7.1 Mechanisms of leukocyte infiltration in tissues – Extravasation.................19
1.2.8 Study of adipose tissue and adipocytes ..........................................................22
1.2.9 Study of endothelial cells in vitro ..................................................................24
1.2.10 Study of cross-talk between cell types ...........................................................25

1.3 Aim of the study.........................................................................................27
2 MATERIALS AND METHODS..................................................... 29

2.1 Cell Culture................................................................................................29
2.1.1 Primary cell culture and size fractionation of human mature adipocytes ........29
2.1.1.1 Reagents....................................................................................................29
2.1.1.2 Theoretical Background and Method .........................................................29
2.1.2 SGBS cell culture ..........................................................................................30
2.1.2.1 Reagents....................................................................................................30
2.1.2.2 Theoretical Background and Method .........................................................31
2.1.3 HMEC-1 cell culture .....................................................................................32
2.1.3.1 Reagents....................................................................................................32
2.1.3.2 Theoretical Background and Method .........................................................33
2.1.4 U937 cell culture ...........................................................................................33
2.1.4.1 Reagents....................................................................................................33
2.1.4.2 Theoretical Background and Method .........................................................33
- i - 2.1.5 Coculture between CM of SGBS cells and HMEC-1 cells .............................34
2.1.6 Coculture and monocyte-endothelial cell-cell adhesion assay ........................34
2.1.6.1 Theoretical Background.............................................................................34
2.1.6.2 Reagents....................................................................................................35
2.1.6.3 Method......................................................................................................35

2.2 Cell surface expression analysis by cell-ELISA........................................36
2.2.1 Theoretical Background.................................................................................36
2.2.2 Reagents........................................................................................................36
2.2.3 Method..........................................................................................................37

2.3 Proliferation assay .....................................................................................38
2.3.1 Theoretical Background.................................................................................38
2.3.2 Reagents........................................................................................................38
2.3.3 Method..........................................................................................................38

2.4 RNA extraction, DNAse I treatment and RNA integrity check...............39
2.4.1 Reagents........................................................................................................39
2.4.2 Theoretical Background and Method .............................................................39

2.5 Reverse transcription (RT), polymerase chain reaction (PCR) and
quantitative real-time PCR (qRT-PCR) ...................................................40
2.5.1 Theoretical background .................................................................................40
2.5.1.1 SYBR® Green...........................................................................................41
2.5.1.2 TaqMan® ..................................................................................................42
2.5.1.3 Primer design ............................................................................................42
2.5.2 RT.................................................................................................................42
2.5.2.1 Reagents....................................................................................................42
2.5.2.2 Method......................................................................................................42
2.5.3 PCR...............................................................................................................43
2.5.3.1 Reagents....................................................................................................43
2.5.3.2 Method......................................................................................................43
2.5.4 qRT-PCR ......................................................................................................44
2.5.4.1 Reagents....................................................................................................44
2.5.4.2 Method......................................................................................................44

2.6 Measurement of adipokines in CM...........................................................45
2.6.1 Theoretical Background.................................................................................45
2.6.1.1 ELISA................................................................................................