Abstract
Background/aim: The transplantation of genetically modified livers or hepatocytes has been discussed as therapeutical option for immunomodulation or treatment of liver function defects. A combination of transfection of the gene carrying plasmid and infection with a Vaccinia-Virus in hepatocytes was investigated for a sufficient gene transfer in primary human hepatocytes. The influence of ...
Abstract
Background/aim: The transplantation of genetically modified livers or hepatocytes has been discussed as therapeutical option for immunomodulation or treatment of liver function defects. A combination of transfection of the gene carrying plasmid and infection with a Vaccinia-Virus in hepatocytes was investigated for a sufficient gene transfer in primary human hepatocytes. The influence of hypothermal storage was evaluated with regard to gene transfer and subsequent gene expression.
Methods: Primary human hepatocytes were isolated from human liver resections using EGTA/collagenase-perfusion. After 24 h of culture gene transfer was performed by infection of cells with a recombinant, replication incompetent vaccinia virus followed by liposome-mediated transfection of the reporter plasmid. The gene products were quntified after infection at 37 degrees C and 4 degrees C up to 8 days.
Results: Gene transfer was successful in 30% of cells as measured by the expression of gfp (green fluorescent protein) as a marker for transinfection. Under hypothermal conditions gene expression was reduced to 20-25%. The expression of HBsAg in the supernatant was measured up to 8 days of culture.
Conclusion: Transinfection with the highly attenuated, replication incompetent vaccinia virus is a valuable model for efficient gene transfer into primary human hepatocytes without major cell damage. Therefore, gene expression of variable plasmids is possible without construction of recombinant vaccinia viral vectors. Organ- or cell preservation at 4 degrees C for potential clinical use in the transplant setting seemes to have no major influence on efficiency and function of gene transfer.
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