Korean J Med.
1999 Jul;57(1):24-35.
Conditional immortalization of human fetal hepatocytes using an amphotropic retrovirus encoding temperature - sensitive SV40 large T antigen
- Affiliations
-
- 1Department of Internal Medicine, Kyung Hee University College of Medicine, Seoul, Korea.
- 2Department of Obstetrics and Gynecology, Kyung Hee University College of Medicine, Seoul, Korea.
- 3Department of General Surgery, Kyung Hee University College of Medicine, Seoul, Korea.
Abstract
-
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.
Keyword
- Full Text Links
-
- Actions
-
Cited
- CITED
-
- Close
- Share
-
- Similar articles
-
- Immortalized hepatocytes established with a temperature-sensitive SV40 T antigen could be independent of T antigen
- Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage
- Effect of Long-term Passage on Liver-specific Functions in Immortalized Hepatocytes with a Temperature-sensitive Mutant of SV40 T Antigen
- Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen
- Establishment of Human Myofibroblast Like Cell Line Immortalized by Transfection of SV40 Largw T Gene
