Morphological characterization of liver fibrogenesis in animal models with genetically modulated {TGF-β [TGF-beta] signal transduction [Elektronische Ressource] / vorgelegt von Jafar Hamzavi Sarkhaei

Borsu Schekuhmand

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2007
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 10 Mo

Extrait

Morphological characterization of liver fibrogenesis in animal models with
genetically modulated TGF- β signal transduction




Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-Westfälischen
Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der
Naturwissenschaften genehmigte Dissertation

vorgelegt von

Diplom-Biologe
Jafar Hamzavi Sarkhaei
aus Abadan im Iran


Berichter: Universitätsprofessor Dr. rer. nat. S. Dooley
Universitätsprofessor Dr. rer. nat. F. Kreuzaler


Tag der mündlichen Prüfung: 08. Juni 2007

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar
Table of contents


Abbreviations ........................................................................................................................................................................................................... 7
1 Introduction.................................................................................................................................................................................................. 8
1.1 Liver function .............................................................................................................................................................................................. 8
1.2 Liver fibrosis................................................................................................................................................................................................ 9
1.3 Hepatic stellate cells .................................................................................................................................................................................... 9
1.4 Hepatic stellate cell activation ................................................................................................................................................................... 10
1.5 TGF- β in tissue repair................................................................................................................................................................................ 11
1.6 TGF- β in fibrogenesis................................................................................................................................................................................ 13
1.7 TGF- β/Smad signal transduction............................................................................................................................................................... 14
1.8 Animal models for liver disease ................................................................................................................................................................ 17
1.8.1 Induction of liver damage with dimethylnitrosamine ................................................................................................................................ 17
1.8.2 Induction of liver fibrosis with carbon tetrachloride.................................................................................................................................. 18
1.8.3 Establishing liver damage with bile duct ligation ...................................................................................................................................... 18
1.9 Blunting fibrogenesis by blocking TGF- β ................................................................................................................................................. 18
1.9.1 Anti-TGF- β antibodies.............................................................................................................................................................................. 19
1.9.2 Dominant-negative and soluble TGF- β type II receptors.......................................................................................................................... 19
1.9.3 Smad7 ........................................................................................................................................................................................................ 20
1.9.4 Compound drugs to inhibit profibrogenic TGF- β signaling ..................................................................................................................... 21
1.10 C-reactive protein in liver damage............................................................................................................................................................. 23
1.11 Aim of the study ........................................................................................................................................................................................ 25
2 Materials and Methods............................................................................................................................................................................... 26
2.1 Materials .................................................................................................................................................................................................... 26
2.1.1 General chemicals...................................................................................................................................................................................... 26
2.1.2 Antibodies.................................................................................................................................................................................................. 27
2.1.3 Reagents and material for animal experiments .......................................................................................................................................... 28
2.1.4 Reagents for immunhistochemistry............................................................................................................................................................ 29
2.1.5 Reagents for preparation of protein lysates................................................................................................................................................ 29
2.1.6 Protocol for RIPA buffer preparation ........................................................................................................................................................ 30
2.2 Animals...................................................................................................................................................................................................... 30
2.2.1 Origin of the FVB/N strain ....................................................................................................................................................................... 30
2.2.2 Origin of the CD-1 strain ........................................................................................................................................................................... 31
2.3 Bacteria...................................................................................................................................................................................................... 32
2.4 Plasmids..................................................................................................................................................................................................... 33
2.5 Cloning strategy to create CRP-Smad7 ..................................................................................................................................................... 35 Table of contents

2.5.1 Amplification of plasmids pcDNA3.1-Flag-Smad7 and U2 ...................................................................................................................... 35
2.5.2 Preparation of bacterial preparatory cultures ............................................................................................................................................. 36
2.5.3 Growth of bacteria ..................................................................................................................................................................................... 36
2.5.4 DNA plasmid maxi preparation ................................................................................................................................................................. 36
2.5.5 Measuring plasmid DNA concentration..................................................................................................................................................... 36
2.5.6 Plasmid restriction digestion analysis ........................................................................................................................................................ 37
2.5.7 Digestion of plasmid pcDNA3.1-Flag-Smad7........................................................................................................................................... 37
2.5.8 Digestion of plasmid U2 ............................................................................................................................................................................ 38
2.5.9 DNA agarose gel electrophoresis............................................................................................................................................................... 38
2.5.10 Dephosphorylation of DNA fragments ..............................................