Induzierter Zelltod von transdifferenzierten hepatischen Sternzellen (Myofibroblasten) auf der Basis des Thymidinkinase-Ganciclovir-Systems

In the current study the thymidine kinase / ganciclovir-system was used to develop a prophylaxis in oder to prevent liver fibrogenesis. The intention was to eliminate activated hepatic stellate cells (HSC), those cells which became proliferative, fibrogenetic and contractile because of liver injury. Activated HSC are called myofibroblasts. They are mainly responsible for the expression of extracellular matrix protein leading to excessive scar formation and finally to cirrhosis, the end stage of fibrosis. Therefore the herpes simplex thymidinikinase gene was coupled to the human tissue inhibitor of metalloproteinases-1-promoter (h-TIMP-1-promoter), which is known as a strongly activated promoter within progressive liver fibrosis. Shuttle vector and recombinant replication-defective adonovirus was generated, containing the h-TIMP-1 / thymidine kinase transgene. After transfection of the plasmid into HSC and after infection of HSC with an adenovirus a strong expression of thymidine kinase-mRNA was observed in the cells. Furthermore protein synthesis of thymidinkinase and its correct enymatic activity was proven. The infected cells were able to modify the prodrugs aciclovir and ganciclovir into aciclovir- and ganciclovirtriphosphat. The toxicity of ganciclovir was determind via dilution series in cell culture. A successful treatment with the combination of thymidine kinase and ganciclovir in cells was observed and could be reproduced. The treatment caused cell death. It was shown that single components are not toxic to the cells. Furthermore, a Bystander Effect was observed. It became clear, that the activity of the h-TIMP-1-promoter is different depending on the celltype: under controll of the h-TIMP-1-promoter thymidine kinase was strongly expressed in HSC and less in myofibroblasts. To analyze the level of expression in quiescent HSC experiments were carried out in teflon tubes in order to to keep the cells as non-activated HSC. The results directed to the supposition that h-TIMP-1-promoter isnt active in quiescent HSC. The induced cell death was identified as apoptosis; necrosis could be excluded. For further animal experiments with rats a detailled plan was established. The effect of liver fibrosis as a consequence of bile duct ligation was qualified and quantified by monitoring the animals, analysing laboratory parameters and characterising tissue samples. It was not possible to make a certain conclusion about the effect of thymidine kinase / ganciclovir-system and the “bystander effect” in vivo.