Reversibly immortalized hepatic progenitor cell line containing double suicide genes

A large number of functional hepatocytes is required for bioartificial liver therapy. Simian virus 40 T‑antigen (SV40T) has been previously reported to improve the immortalized proliferation of primary hepatocytes to generate a sufficient number of cells; however, these long‑term immortalized hepatocytes may induce further malignant transformation in vivo. In the present study, the SV40T immortalization gene and two suicide genes, herpes simplex virus thymidine kinase (HSV‑tk) and cytosine deaminase (CD), were transducted into primary hepatocytes to construct a novel type of Cre/LoxP‑mediated reversible immortalized hepatocyte line. Polymerase chain reaction analysis and western blotting confirmed that the SV40T, HSV‑tk and CD genes were successfully inserted into hepatic progenitor cells and their expression was controlled by Cre/LoxP recombination. Total removal of SV40T could be achieved via the ganciclovir (GCV)/HSV‑tk suicide system. Cells maintained their biosafety in vivo with CD gene expression and 5‑fluorocytosine (5‑FC) induced cell death. Following transplantation into the carbon tetrachloride (CCl4) model group, the majority of cells had survived after 14 days post‑implantation and a number of the cells had transported into the liver parenchyma. When compared with the CCl4 model group, the transplanted cells repaired the liver biochemical index and pathological structure markedly. Thus, the present study reports a novel reversible immortalized hepatocyte with double suicide genes, which exhibited the cellular phenotype and recovery function of normal liver cells. This method maximally guaranteed the biological safety of immortalized hepatocytes for in vivo application, providing a reliable, safe and ideal cell material for the artificial liver technique.