Conditional immortalization of human fetal hepatocytes using an amphotropic retrovirus encoding temperature - sensitive SV40 large T antigen / 대한내과학회지

BACKGROUND: Human cells are almost never spontaneously immortalized in vitro. We tried to immortalize human fetal hepatocytes (h-FH) and evaluate the differentiational status and its change. METHODS: Hepatocytes were isolated from a liver fragment of 20 week old fetus and infected with amphotropic recombinant retrovirus containing a temperature- sensitive mutant of SV40 large T antigen and neomycin phosphotransferase gene. G418 resistant colonies were cloned and expanded. The cells which were able to divide more than 30 times were used to analyze various functions. RESULTS: The immortalization rate was 3.3 x 10-8 and two cell lines (C11, D21) were established. C11-60, C11-80, D21-30 and D21-60 (suffix number means the cell division counts) were evaluated. D21-30 was thougt to be imcompletely immortalized because a considerable portion of cells died during culture. The morphology was similar to that of epithelial cells except for D21-30 which looked like fibroblast. The cells grew rapidly at 33oC but stopped growing at 39oC. T antigen and p53 was expressed at 33oC but disappeared at 39oC, which suggest that T antigen binds to p53. Chromosomal changes were so marked that it was impossible to discriminate exact number. Albumin was secreted as about 1/10 as that of h-FH, but alpha-fetoprotein secretion stopped after immortalization. Telomerase was activated in both cell lines except for the incompletely immortalized cells D21-30. Telomere was elongated in competely immortalized cell lines, but it was rather shortened in D21-30 compared to that of h-FH. Macroscopic colonies did not develop in soft agar assay. CONCLUSIONS: We successfully immortalized human fetal hepatocytes. Although the cells are not likely to have oncogenicity, the functions are not so good, possibly due to marked chromosomal changes which are thought to occur before telomerase is activated during immortalization step.

Study on PA317 Producing High Titers of Retroviral Vector Inserted with Human TNF-?Gene / 中国肿瘤生物治疗杂志

In order to use retroviral-mediated gene transfer technology in clinical application, retroviral vector must be of high titer and free of detectable replication-competent retroviruses (RCR). The aim of this study was to optimize methods of defective retroviral vector production. Study was conducted using a LXSN vector inserted with human tumor necrosis factor-? gene and an amphotropic retrovirus packaging cell line-PA317. The results indicated that viral titer was influenced by volume of medium and concentration of fetal calf serum. Inactivation of retroviral vector was greater at 37癈 than at 32癈. In experiment of transfection of PA317 and transduction of 3T3, integration of retroviral vector into genome of packaging cells and target cells, and free of RCR were detected by polymerase chain reaction analysis. Viral vector with high titer and free of RCR is able to use in clinical trial