Leber-Fibrose: Erstellung eines Modells zur Analyse lebertoxischer Substanzen und Analyse der ltbp1-abhängigen Aktivierung hepatischer Sternzellen

The liver is the central organ of metabolism and at the same time constitutes the biggest gland of the mammalian organism. Breakdown of liver function after chronic damage caused by viral infection or toxic substances implicates severe consequences for the organism. Research on the causes and the pathogenesis of liver diseases therefore is of major importance. This work adresses two questions in relation to liver fibrosis. The first part of this work deals with the expression of EGFP as a transgenic hepatocyte specific surface marker, by means of which hepatic precursors can be isolated from in vitro differentiated murine embryonic stem cells to generate functional cells for toxicological screenings. After validation of the isolation method (MACS), a reporter construct with an albumin promotor driven expression of the surface-/selection-marker was cloned and the expression specificity was analysed in vitro and in vivo. The functional expression of the transgene and its correct subcellular localization was confirmed using the hepatoma cell line HEPA-1-6. However, transfected ES cells showed basal, albumin independent expression of the transgene and even after in vitro differentiation into hepatocyte like cells did not express sufficient amounts of EGFP protein. Analysis of a transgenic mouse model generated by oocyte injection with the same reporter construct revealed that even under in vivo conditions only marginal amounts of EGFP were produced in the liver. Using a targeting strategy which aimed to knock in the EGFP-selection marker into the mouse albumin locus did not lead to the identification of positive ES cell clones. In the second part of this work, the influence of the latent transforming growth factor beta binding protein 1 (LTBP1) on the „activation“ of hepatic stellate cells (HSC) was analysed. Following „activation“ HSC transdifferentiate towards a myofibroblastic phenotype and constitute the most important fibrogenic cell type during the pathogenesis of liver fibrosis. Using a ltbp1-deficient mouse model in which both known protein isoforms LTBP1-L and LTBP1-S are missing, revealed a new ltbp1 splice variant of which analogous isoforms also exist in rats and humans. Microarray analysis was used to identify known and so far unknown marker genes for activated HSC and more importantly demonstrated that ltbp1-deficient HSC have a reduced tendency to transdifferentiate in vitro. This observation was independently confirmed in vivo using an experimental model for the induction of liver fibrosis (ligation of the common bile duct). After 4 weeks of bile duct obstruction, ltbp1-/- mice showed markedly reduced signs of liver fibrosis, i.e. less Collagen I deposition by HSC in the affected liver parenchyma. The study therefore provides insight into the role of LTBP1 during the pathogenesis of liver fibrosis and points out the clinical relevance of LTBP1, which needs to be confirmed by subsequent analysis.