Establishment And Characterization Of Immortalized Porcine Hepatocyte Lines By Transfection With SV40 Large T Antigen And Human Telomerase Reverse Transcriptase

IntroductionConsidering the potential of the liver to regenerate, Liver support system is urgently needed for patients with FHF as a bridge to transplantation or avoiding the transplantation. Many researchers have made great efforts to develop bioartificial liver (BAL) using various types of cells in different modules. Porcine hepatocytes are considered to be the best candidate for use in a bioartificial liver. To date, various hybrid artificial liver support systems (HALSS) using primary porcine hepatocyte have been submitted for clinical trials.In addition to Primary porcine hepatocytes are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices, Primary cultures of hepatocytes have been used as a model system to investigate liver function, including drug metabolism, hepatotoxicity, protein biosynthesis and gene expression. However, primary cultures of hepatocytes are difficult to proliferate and have a short lifespan in vitro, and lose phenotypic expression and total P450 proteins in culture. So, it is important to find a way of extending their replicative potential.In this study, we explored to establish immortalized porcine hepatoctye lines, retrovirus-mediated gene transfer of SV40 large T antigen and Human telomerase reverse transcriptase, which could be expanded in culture for use as a model system. Characteristics of immortalized porcine hepatocyte lines were evaluated by reverse transcription polymerase chain reactions (RT-PCR), Western blot analysis, tumorigenicity tests in nude mice, and so on.Materials and Methods1. Construct retroviral vectors containing hTERT or SV40 LT and produce retrovirusPT67 retroviral packing cells were transfected with the recombinant retroviral constructs (pl_XSN-SV40 LT and pLPCX-hTERT) by standardized calcium phosphate method, respectively. Both neomycin resistant cells and puromycin resistant cells were selected and subcultured. Viral titers were estimated by NIH 3T3 cells analysis, respectively. The supernatant, containing the recombinant retrovirus, was harvested, filtered through a 0.45 um filter, and frozen at -70℃ until its use as the source of infection for immortalization.2. Immortalization of porcine Hepatocytesprimary porcine hepatocytes were infected with a 100 ul viral stock per plate in the presence of 8 ug/ml polybrene at 37 ℃ for 24 h. Neomycin resistant hepatocytes were selected by adding the neomycin analogue G418 at 400 mg/ml. G418-resistant colonies were isolated using the cloning cylinder, and subcultured by trypsinization. When cultures became 70% confluent, the cells were again infected with 100 ul of another viral stock per plate in the presence of 8 ug/ml polybrene at 37 ℃ for 24 h. Puromycin resistant hepatocytes were selected, isolated and expanded by subculture for further studies.3. Characterization of immortalized porcine hepatocytesCharacteristics of immortalized porcine hepatocyte lines were evaluated by reverse transcription polymerase chain reactions (RT-PCR), Western blot analysis, tumorigenicity tests in nude mice, scanning electron microscopy (SEM), transmission electron microscopy (TEM), enzyme-linked immunosorbent assay, and so on.Results1. Construct retroviral vectors containing hTERT or SV40 LT and produce retrovirusRetroviral vector containing hTERT or SV40 LT was constructed by DNA recombinant techniques in vitro. The combinant vector was determined with enzyme digestion and sequencing and was transfected into the PT67 cell lines by standardized calcium phosphate method and screened by G418 or Puromycin. Anti-G418 or anti-Puromycin positive clones were established. The viral titer was determined with the NIH3T3.2. Establishment of the Immortalized porcine Hepatocyte LinesPrimary porcine hepatocytes were transfected with the recombinant retroviral vectors by exposure to amphotropic retroviral supematants. The transfected clones grew within 4 weeks, whereas nontransfected primary porcine hepatocyte died within 2 weeks. After 6 months, all three transfected clones were still proliferating. One of them, referred to as hepLP, was composed of cells having a large round nucleus with a few nucleoli and many granules in the cytoplasm under a phase-contrast microscope (PCM), which are characteristics of primary porcine hepatocytes. The result of SEM and TEM showed that HepLP cells maintained a round nucleus with nucleoli, moderate numbers of mitochondria, and numerous rough endoplasmic reticulums.3. Characterization of the Immortalized porcine Hepatocyte LinesThe protein expression of SV40 LT and hTERT was detected by Western blot in all the Immortalized porcine Hepatocyte Lines. We analyzed the growth kinetics of HepLP cells by standard culture conditions; it showed that the number of HepLP cells was significantly increased in all periods of observation. In contrast, the primary cultured hepatocytes did not apparently proliferate during a 5-day period. Up to date, the cells were maintained for more than 60 passages with an average of one population doubling every 6-8 hours. The concentration of porcine albumin in the supernant was higher in HepLP cells than that in primary cells. The capacity for urea synthesis of HepLP cells was stronger than that in primary hepatocyte. The result of RT-PCR showed that HepLP cells expressed albumin mRNA as well as primary hepatocyte.When the nude mice were injected subcutaneously with 1×10~6 HepLP cells, they did not develop any tumor for as long as 6 months after injection, whereas nude mice with transplantation of the same amount of HepG2 cells developed tumors in 3-4 week after injection.Conclusions1. Retroviral vector containing hTERT or SV40 LT was constructed by DNA recombinant techniques in vitro.2. Two high titer toxigenous cell line contained hTERT or SV40 large T antigen gene were established successfully.3. The immortalized, nontumorigenic, porcine hepatocytes maintained the properties of porcine primary hepatocytes such as the albumin secretion. This generation of immortalized porcine hepatocyte may be helpful for bioartifical liver support system, hepatocytes transplantation, drug/toxicological studies, and liver biologic studies.