TGF-β,Smad [TGF-beta,Smad] signaling in hepatic stellate cells and during liver fibrogenesis [Elektronische Ressource] / vorgelegt von Eliza Wiercinska

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

"TGF-β/Smad signaling in hepatic stellate cells and
during liver fibrogenesis"
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
einer Doktorin der Naturwissenschaften genehmigte Dissertation
vorgelegt von
Diplom-Chemikerin
Eliza Wiercinska
aus Gdansk in Polen
Berichter: Universitätsprofessor Dr. Steven Dooley
Universitätsprofessor Dr. Fritz M. Kreuzaler
Tag der mündlichen Prüfung: 15. Dezember 2005
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.To my parents1. Summary............................................................................................................................... 5
1a. Zusammenfassung.............................................................................................................. 6
2. Introduction .......................................................................................................................... 7
2.1. Liver fibrogenesis....................................................................................................................................... 7
2.2. TGF- β signal transduction pathways ...................................................................................................... 9
2.2.1. Smad proteins...................................................................................................................................... 11
2.2.2. Smad7 ................................................................................................................................................. 13
2.3. The basic Helix-Loop-Helix transcription factor family....................................................................... 15
2.3.1. Id1 ....................................................................................................................................................... 16
2.4. Anti TGF-β strategies for the treatment of fibrosis (based on Breitkopf et al., in press). ................. 18
2.5. Aim of the study ....................................................................................................................................... 20
3. Materials and Methods ...................................................................................................... 21
3.1 Buffers, media, markers and chemical reagents..................................................................................... 21
3.1.1. Cell culture reagents............................................................................................................................ 21
3.1.2. Cytokines ............................................................................................................................................ 22
3.1.3. Buffers for Western blot...................................................................................................................... 22
3.1.4. Buffers for immunfluorescent staining of monolayer cell culture....................................................... 23
3.2. Eukaryotic cells and their culture........................................................................................................... 24
3.2.1. Cells .................................................................................................................................................... 24
3.2.2. Cell culture.......................................................................................................................................... 24
3.3. Adenoviruses............................................................................................................................................. 25
3.3.1. Virus amplification ............................................................................................................................. 25
3.3.2. Constructs ........................................................................................................................................... 25
3.3.3. Infection of cell cultures...................................................................................................................... 27
3.3.4. Determination of virus titer ................................................................................................................. 27
3.3.5. Determination of virus functionality ................................................................................................... 28
3.3.6. Testing of cell vitality after virus infection ......................................................................................... 29
3.4. Animal experiments ................................................................................................................................. 30
3.4.1. Blood tests........................................................................................................................................... 31
3.4.2. Tissue samples .................................................................................................................................... 31
3.4.3. Hydroxyproline assay.......................................................................................................................... 31
3.5. Western blot.............................................................................................................................................. 33
3.5.1. Protein lysates ..................................................................................................................................... 33
3.5.2. Protein concentration .......................................................................................................................... 33
3.5.3. Preparation of samples for western blot .............................................................................................. 33
3.5.4. SDS – polyacrylamide gel electrophoresis.......................................................................................... 33
3.5.5. Western transfer .................................................................................................................................. 33
3.5.6. Antibodies ........................................................................................................................................... 34
3.5.7. Immunodetection of proteins .............................................................................................................. 35
3.6. Immunostaining of monolayer cells........................................................................................................ 35
33.6.1. Preparation of glass slides and cell culture ......................................................................................... 35
3.6.2. Staining ............................................................................................................................................... 35
3.7. Detection of mRNA expression ............................................................................................................... 36
3.7.1. RNA isolation from monolayer cells................................................................................................... 36
3.7.2. RNA electrophoresis ........................................................................................................................... 36
3.7.3. Reverse transcription........................................................................................................................... 37
3.7.4. Polymerase chain reaction (PCR) ....................................................................................................... 37
3.7.5. Real-time PCR quantification ............................................................................................................. 37
3.8. Promoter-reporter assays........................................................................................................................ 38
3.8.1. Transfection of monolayer cells.......................................................................................................... 38
3.8.2. Luciferase assay .................................................................................................................................. 38
4. Results (based on (Dooley et al., 2003) and (Wiercinska et al., submitted)) ................. 39
4.1. Expression of the Smad7 transgene in rat liver..................................................................................... 39
4.2. Effect of Smad7 expression on collagen synthesis and activation of HSC in liver fibrogenesis ........ 40
4.3. Ectopic expression of Smad7 inhibits HSC activation .......................................................................... 45
4.4. Ectmad7 blocks paracrine and autocrine TGF-β signal transduction in HSC . 46
4.5. Effects of Smad7 on α-SMA expression during culture activation of HSC......................................... 49
4.6. Smad7 interferes with Id1 expression in activated HSC....................................................................... 51
4.7. Ectopic Id1 expression enhances activation of HSC and abrogates inhibitory effects of Smad7...... 52
4.8. RNA interference-mediated knock down of Id1 expression inhibits transdifferentiation of HSC ... 54
4.9. TGF-β induces expression of Id1 in HSC via the ALK1 but not the ALK5 pathway ........................ 56
4.10. Id1 protein expression is upregulated during activation of HSC...........................