Publikationsserver der RWTH Aachen University
Einfluss von Transforming Growth Factor-beta1 (TGF-[beta]1) Genpolymorphismen auf die Hepatitis-C-induzierte Leberfibrose in vivo und Modulation der TGF-[beta]1-Synthese, Prozessierung und Sekretion durch die Serin-Protease Furin, das latent TGF-[beta] binding protein-1 und Polymorphismen der TGF-[beta]1-Signalsequenz in vitro
Abstract
dc:descriptionIn the first part of this thesis, the influence of TGF-beta1 polymorphisms at position –509, codon 10, codon 25 and codon 263 on hepatitis C induced liver fibrosis was analysed. For that purpose, liver needle biopsies from 430 patients suffering from chronic hepatitis C infection were scored for the necroinflammatory activity (grading) and degree of hepatic fibrosis (staging). Protocols allowing the isolation of genomic DNA from respective paraffin embedded tissues were optimized and novel LightCycler-genotyping methods for respective allelic variants were established. Subsequently, gender and polymorphisms were statistically correlated with grade and stage of respective samples. In addition, it was tested, if the allele frequencies between patients who suffer from mild or severe fibrosis were different. The data showed a statistically significant association between gender and stage as well as codon 10 and staging. This analysis revealed a significant difference in the allelic frequences at position –509 between the patients suffering from mild or severe fibrosis. Patients being CT or TT at postion –509 had lower stage than patients being CC at this point. There was no correlation of allelic variations and necroinflammatory activity. A significant linkage between the polymorphism at position –509 and the polymorphisms at codon 10 and codon 263 was also found in this analysis. Furthermore, the examinations showed a significant association between the polymorphisms at codon 10 and codon 25. In the second part of the thesis, the influence of the furin convertase, the latent TGF-beta binding protein-1 (LTBP-1) and the individual polymorphisms at codon 10 and codon 25 on the expression and cellular secretion of TGF-beta1 was analysed in vitro. To do so, human TGF-beta1 expression constructs of the allelic variations 10Leu/25Arg, 10Pro/25Pro and 10Pro/25Arg were cloned into a C-terminal Flag tagging vector harbouring the CMV promotor. These vectors were then transfected alone or in combination with expression vectors for furin and LTBP-1 into CHO cells or into LX-2 cells. The expression and secretion was confirmed by Western blot and quantification of Flag-eptitope tagged protein was done in ELISA. In the two cell systems, synthesis and proteolytic cleavage of TGF-beta1 were influenced differently by furin and LTBP-1. In CHO cells, furin and/or LTBP-1 co-expression resulted in an increase of TGF-beta1 in both cell lysate and supernatant. In contrast, the TGF-beta1 synthesis in LX-2 cells was suppressed by furin and LTBP-1. The TGF-beta1 polymorphisms also influenced the total TGF-beta1 concentration. The allelic variations 10Pro/25Arg and 10Pro/25Pro showed a significant lower TGF-beta1 concentration than the 10Leu/25Arg variant. Thus, the experiments showed that the non-synonymous nucleotide substitutions at codon 10 and codon 25 suppress the TGF-beta1 synthesis. Furthermore, a reporter vector in which human TGF-beta1 and the enhanced green fluorescent protein were fused (hTGF-b1-EGFP) was established and the cellular processing of TGF-beta1 was analysed in confocal laser scanning microscopy. The expression of TGF-beta1-EGFP fusion showed that TGF-beta1 is secreted in distinct cytosolic bodies.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2008
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Mohren, Simone
- Contributors dc:contributor
-
- Weiskirchen, Ralf
Subjects
dc:subject × 11Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger