Molecular and functional characterization of murine macrophage subtypes [Elektronische Ressource] / vorgelegt von Daniela Dreymüller

Danielaalt

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2009
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 22 Mo

Extrait

“Molecular and functional characterization of murine
macrophage subtypes”
Von der Fakultät für Mathematik, Informatik und
Naturwissenschaften der RWTH Aachen University zur
Erlangung des akademischen Grades einer Doktorin der
Naturwissenschaften genehmigte Dissertation
vorgelegt von
Diplom-Biologin Daniela Dreymüller
aus Aachen
Berichter:
Universitätsprofessor Dr. Wilhelm Jahnen-Dechent
Universitätsprofessor Dr. Lothar Elling
Tag der mündlichen Prüfung: 16.06.2009
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek
online verfügbar.Content I
Summary................................................................................................................................1
1. Introduction ............................................................................................................3
1.1. Macrophage biology ...............................................................................................3
1.1.1. Murine macrophage subtypes..............................................................................3
1.1.1.1. Murine macrophage subtypes from embryo to newborn...................................3
1.1.1.2. Macrophage subtypes in adult mice.................................................................4
1.1.1.3. Markers for embryonic/fetal and adult macrophages........................................5
1.1.2. The macrophage microenvironment ....................................................................6
1.1.2.1. The extracellular matrix – macrophage interplay .............................................6
1.1.2.2. Macrophage activation states...........................................................................7
1.1.3. Macrophage function ..........................................................................................8
1.1.3.1. Endocytosis.....................................................................................................9
1.1.3.2. Lysosomal degradation..................................................................................11
1.1.3.3. Antigen presentation .....................................................................................12
1.1.3.4. Migration ......................................................................................................13
1.2. Wound healing......................................................................................................14
1.2.1. Wound healing in the embryo/fetus...................................................................16
1.2.2. Wound healing and macrophages – inflammation and scaring...........................16
1.2.3. Models for wound healing.................................................................................18
1.3. Macrophage model ...............................................................................................18
1.4. Experimental aims ................................................................................................20
2. Material and Methods ...........................................................................................21
2.1. Cell culture ...........................................................................................................21
2.1.1. Murine feeder cell culture .................................................................................21
2.1.2. R1 cell culture...................................................................................................21
2.1.3. ES macrophage differentiation and culture ........................................................21
2.1.4. BM macrophage differentiation and culture.......................................................22
2.1.5. Saphenous vein endothelial cell (SVEC) culture................................................22
2.1.6. Rat hepatoma cell line FTO2B culture...............................................................22
2.2. Determination of cell size .....................................................................................22
2.3. Activation profiling...............................................................................................23
2.3.1. Stimulation by LPS ...........................................................................................23
2.3.2. Oxidative burst measurement ............................................................................23
2.3.3. Classical and alternative activation....................................................................23II Content
2.3.3.1. Expression of marker genes CD163, CD206 and CCL3.................................23
2.3.3.2. Cytokine expression profile...........................................................................23
2.3.4. Leukocyte acidic phosphatase activity...............................................................24
2.3.5. Lysozyme activity.............................................................................................24
2.3.6. Osteoclast differentiation...................................................................................24
2.4. Surface marker profiling .......................................................................................24
2.5. Analysis of macrophage function ..........................................................................25
2.5.1. Endocytosis.......................................................................................................25
2.5.1.1. Receptor-mediated endocytosis .....................................................................25
2.5.1.2. Pinocytosis....................................................................................................26
2.5.1.3. Pinocytosis and phagocytosis of latex beads..................................................26
2.5.1.4. Phagocytosis of apoptotic cells......................................................................26
2.5.1.5. Phagocytosis of rat hepatoma cells (F2OTB cells)........................................26
2.5.1.6. Phagocytosis of bioparticles ..........................................................................26
2.5.1.7. Endocytosis of bovine fetuin-A monomer and calciprotein particles (CPPs) ..27
2.5.2. Adhesion assays................................................................................................27
2.5.2.1. Coated glass slides ........................................................................................27
TM2.5.2.2. MSA cell adhesion assay ...........................................................................27
2.5.3. Matrix metalloproteinase activity ......................................................................27
2.5.4. T cell activation assays......................................................................................28
2.5.4.1. OT-II proliferation assay ...............................................................................28
2.5.4.2. OT-I proliferation assay ................................................................................28
2.5.5. Transmigration assay ........................................................................................29
2.6. Generation of transgenic E. coli strains .................................................................29
2.6.1. Generation of calcium competent bacteria.........................................................29
2.6.2. Cloning of PCR fragments ................................................................................29
2.6.2.1. Vector-insert-ligation ....................................................................................29
2.6.2.2. Transformation of bacteria ............................................................................29
2.6.2.3. Plasmid preparation.......................................................................................30
2.7. Analysis of gene expression ..................................................................................30
2.7.1. Semiquantitative RT-PCR.................................................................................30
2.7.2. RealTime-PCR..................................................................................................30
2.7.3. Primer systems..................................................................................................31
2.8. In vivo-tail wound model.......................................................................................31Content III
2.8.1. Wounding and cell application ..........................................................................31
2.8.2. Histology and immunhistochemical staining procedure .....................................31
2.8.3. Statistical analysis.............................................................................................33
3. Results..................................................................................................................34
3.1. Determination of cell size......................................................................................34
3.2. Surface marker profiling .......................................................................................34
3.3. Developmental classification.................................................................................35
3.4. Activation profiling...............................................................................................38
3.4.1. Activation by LPS treatment .............................................................................38
3.4.2. Oxidative burst measurement ..........