Transcriptomic profiling and regulatory pathway modeling in a renal allograft transplantation model [Elektronische Ressource] / Christine von Törne

Christine Von Törne

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 28 Mo

Extrait

Transcriptomic profiling and regulatory
pathway modeling in a renal
allograft transplantation model





Dissertation der Fakultät für Biologie der
Ludwig-Maximilians-Universität München

Christine von Törne
München
2009























Erstgutachter: Prof. Dr. Elisabeth Weiß
Zweitgutachter: Prof. Dr. Peter Becker

Tag der mündlichen Prüfung: 19.11.2009
Die vorliegende Arbeit wurde unter der Anleitung von PD Dr. Peter Nelson im
Forschungslabor der Medizinischen Poliklinik der Ludwig-Maximilians-Universität
München durchgeführt.
Das Projekt wurde gefördert durch das Graduierten Kolleg 438 „Vaskuläre Biologie“
der LMU und durch die Deutsche Forschungsgemeinschaft im Rahmen des
Sonderforschungsbereiches 571 ”Autoimmunreaktionen: von den Manifestationen
über die Mechanismen zur Therapie”.

































Table of contents
Table of contents
Abstract...................................................................................................1
Zusammenfassung.................................................................................2
1 Introduction ................................................................................3
1.1 Chronic allograft dysfunction (CAD) ..............................................................3
1.1.1 Inflammation and fibrosis in CAD ...........................................................4
1.1.2 Myofibroblasts ........................................................................................4
1.1.3 Pathways activated in epithelial to mesenchymal transition (EMT) ........5
1.1.3.1 The TGFB pathway.........................................................................6
1.1.3.2 The NOTCH pathway......................................................................7
1.1.3.3 The hedgehog (HH) pathway ..........................................................8
1.1.3.4 The WNT pathway ........................................................................10
1.2 Preceding experiments12
1.2.1 CAD in a Fisher to Lewis rat model......................................................12
1.2.2 Effects of retinoic acid treatment13
1.2.3 Working mechanism of retinoic acid.....................................................14
2 Aim of the present study.........................................................17
2.1 Transcriptomic profiling of kidney transplants..............................................17
3 Material......................................................................................18
3.1 Microarrays..................................................................................................18
3.1.1 CEL files...............................................................................................19
3.2 Kidney samples ...........................................................................................20
3.2.1 RNA isolated from kidney sections.......................................................20
3.2.2 Kidney sections for immunohistochemistry ..........................................20
3.3 Cells ............................................................................................................20
3.3.1 Human fibroblast cell line K4IM............................................................20
3.3.2 Media used for K4IM ............................................................................21
3.4 Oligonucleotides..........................................................................................21
3.4.1 Oligonucleotides for qPCR of cDNA.....................................................21
3.4.1.1 AoD Assays ..................................................................................21
3.4.1.2 QuantiTect Primer Assays21
3.4.1.3 Oligonucleotides for qPCR, self-designed.....................................21
3.4.2 Oligonucleotides for PCR of genomic DNA, self designed...................22
3.5 Enzymes, enzyme solutions, and inhibitors.................................................22
3.6 Antibodies....................................................................................................22
3.6.1 Primary antibodies................................................................................22
3.6.2 Secondary antibodies...........................................................................22
3.7 Kits ..............................................................................................................22
3.8 Chemicals, reagents, and additives.............................................................23
3.9 Buffers and Solutions ..................................................................................24
3.10 Consumables ..............................................................................................25
3.11 Instruments..................................................................................................26
3.12 Software ......................................................................................................27
Table of contents
3.12.1 Commercial software............................................................................27
3.12.2 Freeware and databases......................................................................27
4 Methods ....................................................................................28
4.1 Microarray analysis .....................................................................................28
4.1.1 ChipInspector analysis of CEL Files.....................................................28
4.1.2 BiblioSphere and GO analysis .............................................................29
4.1.3 Pathway analysis .................................................................................29
4.1.4 Robust multichip average (RMA) analysis............................................30
4.1.5 Cluster analysis30
4.2 Verification of microarray results on the mRNA level ..................................31
4.2.1 RNA Cleanup .......................................................................................31
4.2.2 Quantification of RNA...........................................................................31
4.2.3 Reverse Transcription ..........................................................................31
4.2.4 Quantitative PCR (qPCR).....................................................................32
4.2.5 Calculation of regulation.......................................................................33
4.3 Verification of microarray and qPCR results on the protein level.................34
4.3.1 Histology ..............................................................................................34
4.3.2 Immunohistochemistry .........................................................................34
4.3.2.1 Sample preparation35
4.3.2.2 Detection of the antigen ................................................................35
4.4 Identification of the working mechanism of 13cRA on the DNA level ..........37
4.4.1 Cell cultivation ......................................................................................37
4.4.1.1 Counting of viable cells .................................................................37
4.4.1.2 Cryo-conservation of cells.............................................................37
4.4.1.2.1 Freezing of cells.........................................................................37
4.4.1.2.2 Thawing of cells38
4.4.2 Cell culture and stimulation experiments..............................................38
4.4.3 Enzyme-inked immunosorbent assay (ELISA) .....................................39
4.4.4 Chromatin immunoprecipitation (ChIP) ................................................39
4.4.4.1 Chromatin preparation ..................................................................39
4.4.4.1.1 Formaldehyde fixation................................................................39
4.4.4.1.2 Lysis of cells...............................................................................40
4.4.4.1.3 Sonication ..................................................................................40
4.4.4.1.4 Gel electrophoresis ....................................................................40
4.4.4.2 Immunoprecipitation......................................................................41
4.4.4.2.1 Preparation of Pansorbin (StaphA) cells ....................................41
4.4.4.2.2 Blocking of Staphylococcus A cells............................................41
4.4.4.2.3 Preparation of samples ..............................................................41
4.4.4.2.4 Precipitation ...............................................................................42
4.4.4.2.5 Decrosslinking and ethanol precipitation42
4.4.4.2.6 Protease K digestion..................................................................42
4.4.4.3 DNA analysis..........................