The role of Id2 (inhibitor of differentiation/ DNA binding) in dendritic cell development in steady state and inflammation [Elektronische Ressource] / Juliane Luise Ober-Blöbaum

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

“The role of Id2 (inhibitor of differentiation/ bDinNdAing)
in dendritic cell development in steady state and
inflammation”



2., überarbeitete Fassung





Von der Fakultät für Mathematik, Informatiku rwuinds seNnsacthaften der RWTH
Aachen University zur Erlangung des akademisacdhens Geriner Doktorin der
Naturwissenschaften genehmigte Dissertation

vorgelegt von

Bachelor of Science Hono urs
Juliane Luise Ober-Blöbaum

aus Hamburg





Berichter: Universitätsprofessor Dr. Martin Zenke
Universitätsprofessor Dr. Lothar Elling

Tag der mündlichen Prüfung: 08.12.2009


Diese Dissertation ist auf den Internetseitoenc hscdehurl bHibliothek online verfügbar.













Content I

Content
Abbreviations ....................................................... ... .I.V..........
Zusammenfassung ................................................... . .V.I...............
Abstract ......................................................................................... ........ .V.I.I.......
1 Introduction .................................................... .. .1...............
1.1 Basic principles of immunity ................................................................................... 1
1.2 Dendritic cells ....................................................................................................... ...... 3
1.2.1 Migratory dendritic cells ............................................................................................................. 4
1.2.2 Conventional dendritic cells .............................................................................................................. 6
1.2.3 Circulating dendritic cells.............................................................................................................. 7
1.3 Development and activation of dendrit.i..c. ...c..e.l..l..s. ....................................................... 8
1.3.1 Overview of dendritic cell development ................................................................................. 8
1.3.2 Role of cytokines in dendritic cell developm...e..n..t. .................................................................. 1 0
1.3.3 Role of TLRs in dendritic cell activatio.n.. ....................................................................................... 1 2
1.3.4 Role of transcription factors in dendritic ceelollp mdevnt and activation .............................. 1 4
1.4 Aim of the study ..................................................................................................... 19
2 Material and Methods ..........................................2.0. .....................
2.1 Material ....................................................................................................................................0. ..... 2
2.1.1 Chemicals ...................................................................................................................................... 2 0
2.1.2 Size standards ............................................................................................................................. 2 0
2.1.3 Media ............................................................................................................................................ 2 0
Basic culture medium ............................................................................................................... 2 0
Flt3 precursor amplification medium ............................................................................................. 2 0
FL amplification medium .......................................................................................................... 2 0
GM-CSF medium .......................................................................................................................... 2 0
Tissue culture additives: .......................................................................................................... 2 1
2.2 Methods ....................................................................................................................... 24
2.2.1 Mice ................................................................................................................................................ 2 4
Injections ...................................................................................................................................... 2 4
Irradiation ..................................................................................................................................... 2 4
Adoptive transfer ........................................................................................................................ 2 4
UV irradiation ............................................................................................................................. 2 4
2.2.2 Flow Cytometry............................................................................................................................... 2 5
Compensation ............................................................................................................................... 2 5
Cell preparation .......................................................................................................................... 2 5
Intracellular staining .................................................................................................................. 2 5
2.2.3 Isolation of primary cells ........................................................................................................ 2 6
Splenocytes/ thymocytes ................................................................................................................ 2 6
Bone Marrow cells .................................................................................................................... 2 6
Content II

Blood monocytes ........................................................................................................................ 2 6
In vivo labelling of blood monocytes .............................................................................................. 2 6
Skin dendritic cells .................................................................................................................... 2 6
2.2.4 Dendritic cell cultures ................................................................................................................ 2 7
+ +
DC generation from FlCtD311b progenitor cells (two-step cultures) ................................. 2 7
DC generation from GM-CSF bone marrow cultu..r.e..s.. ........................................................... 2 8
DC generation from Flt3L bone marrow cultu.r..e..s. ..................................................................... 2 8
2.2.5 Miscellaneous protocols ............................................................................................................. 2 8
Polymerase chain reaction (PCR) ............................................................................................ 2 8
+
TLR stimulation of DCs and Fpltr3ogenitor cells ..................................................................... 31
Cytometric bead assay (CBA) .................................................................................................. 31
Fluorescence microscopy ................................................................................................................ 31
Confocal microscopy and live cell imaging. ........................................................................... 32
Tissue preparation for transmission electron mcoicpryo s............................................................ 32
Statistics ....................................................................................................................................... 33
Microarray preparation .............................................................................................................. 33
3 Results ..................................................................................... ... .3.4.............
+
3.1 Characterization of an in vitro am plpirfeiceudr soFrl t.3.................................................. 34
+
3.1.1 Surface marker profile of the Fplrte3cursor ............................................................................. 35
+
3.1.2 TLR repertoire of the Flpt3recursor cells ................................................................................ 35
Comparative gene expression analysis for TLR pya thcowmaponents ........................................... 36
+
Expression and up-regulation of TLR mRNA inp rFelctu3rsors and differentiated DCs ....... ...3..8 ....
+
Protein expression of TLRs in Fplrte3cursors and differentiated DCs .................................. 38
+
3.1.3 Functional analysis of TLRs on Fplrte3cursor and differentiated DCs ................................. 40
+
3.1.4 Analysis of the in vivo developmental potentthiea lFoltf3 precursor ....................................... .42
3.2 Development of two distinct types of Lacenglelrsh anisn steady state and inflamm4a6t ion ..........
+
3.2.1 Id2 deficiency results in lack of LCs and ladnDgCesr in...................