engleski

LOW CYP2E1 GENE EXPRESSION PERMITS PHARMACOLOGICAL SELECTION OF TRANSPLANTED HUMAN HEPATOCYTES IN VIVO

To induce proliferation of transplanted human cells in immunocompetent rats by administration of hepatotoxic doses of a selection agent. GFP-Huh7, a hepatocytederived cell line with low CYP2E1 expression, was used. Expression of CYP2E1, an enzyme responsible for conversion of acetaminophen (APAP) into hepatotoxic compounds, was quantitated by real-time PCR. To prevent rejection, immunological tolerance was induced by introduction of GFP-Huh7 cells into fetal rats in utero, prior to development of the immune system. After birth, GFP-Huh7 cells were transplanted and treated with pharmacological doses of APAP. ALT levels and histology were to detect liver damage. GFP (+) cells in rat livers were detected by real-time PCR and fluorescence microscopy. Expression of CYP2E1 in GFP-Huh7 cells was only 4% compared to control cells. Animals treated with APAP increased ALT to 210 IU/ml on day 1, followed by resolution by day 3. Histology confirmed patchy necrosis corresponding in time to the ALT peak. Fluorescence microscopy of livers from transplanted, APAP treated rats showed an increase of GFP (+) cells/field from 4±2 on day 2 to 45±10 on day 7. Real-time PCR confirmed a 10-fold increase of GFP mRNA in APAP treated rats. Control animals had no significant changes of GFP (+) cells or mRNA levels. In vivo, the difference in CYP2E1 gene expression between GFP-Huh7 and host hepatocytes provides a convenient and efficient pharmacological selection resulting in enrichment of GFP-Huh7 cells. Down regulation of CYPs by siRNA may permit application of the selection to other liver cell lines.

humanized animal model; liver transplantation; cytochrome P450-2E1 (CYP2E1); pharmacological selection; acetaminophen (APAP)

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