Samenvatting
Background: Histone deacetylases (HDACs) enzymes are responsible for the deacetylation of chromatin proteins i.e. histones what renders the chromatin less accessible to transcription factors and results in the downregulation of gene expression. HDAC3, one of 18 members of mammalian HDAC family functions as a corepressor for many transcription factors as well as nuclear hormone receptors. HDAC3 was shown to regulate the proliferation, differentiation and apoptosis of cells and metabolic homeostasis of hepatocytes in particular. The aim of this master thesis is to identify the optimal conditions for HDAC3 knock-down using RNA interference technology in cultures of primary rat hepatocytes.Methodology : The primary rat hepatocytes were plated out in a conventional monolayer configuration and cultured in the differentiation-promoting setting for 7 consecutive days. The expression of HDAC3 protein throughout the culture was examined by western blot analysis. In a separate experiment, the hepatoctytes were cultivated in the identical conditions; however, at 4 h or 22 h after plating the cells were transfected with different concentrations of siRNA/PbAE2 polymer complexes. Subsequently, at 46 h and 74 h after the transfection the hepatocytes were harvested and the HDAC3 protein expression was analyzed by immunoblotting, while potential cytotoxic effects of the transfection procedure were monitored by means of lactate dehydrogenase leakage assay and light microscopy observations. Finally, a densitometry analysis of the HDAC3 protein expression with respect to a housekeeping protein (GAPDH) were performed in order to evaluate efficacy of the HDAC3 silencing protocol. Parallel experiments were performed with non-targeting siRNA as a negative control to confirm the specificity of HDAC3 knock-down.
Results : HDAC3 protein demonstrated time-dependent upregulation in the differentiation-promoting culture of primary rat hepatocytes. Upon the transfection of the cells with siRNA/PbAE2 complexes HDAC3 expression decreased in a concentration-dependent manner. Furthermore, the silencing of HDAC3 was more pronounced when the cells were transfected at 22h after plating when compared to 4h after plating. The transfection procedure did not induced adverse effects with respect to cell viability with the exception when 100-1 PbAE2 N-P ratio was used. The "off-target" effects were not observed either.
Conclusion : Our results show that 22h after plating cell transfection and combination of 100 nM siRNA and 75-1 PbAE2 N-P ratio are preferred conditions in order to obtain significant HDAC3 protein downregulation.
| Datum prijs | 2009 |
|---|---|
| Originele taal | Dutch |
| Begeleider | Vera Rogiers (Promotor), Tamara Vanhaecke (Promotor) & Joanna Fraczek (Co-promotor) |