Maintenance of liver function in long term culture of hepatocytes following In Vitro or In Vivo Ha-ras
EJ transfection |
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Authors: | Michel Fischbach Hanwei Cao Miguel Diez Ibanez Christos Tsaconas Sami Alouani Frédéric Montandon Mohammed El Baraka Prudent Padieu Michel Dreano Martine Chessebeuf-Padieu |
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Institution: | (1) Laboratoire de Biochimie Médicale, Faculté de Médecine, UGniversité de Bourgogne, 21033 Dijon;(2) Department of Genetic Engineering, IntraCel SA, CH-1227 Geneva-Carouge;(3) Laboratoire de Biochimie Médicale, Faculté de Médicine, UGniversité de Bourgogne, F-21033 Dijon, France;(4) Ares Serono, CH-1202 Geneva |
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Abstract: | Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-rasEJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 µg albumin and 0.32 µg transferrin per 106 cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminoflourene (2-AFAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHnneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 µg albumin and 11.0 µg transferrin per 106 cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGHper 106 cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 µg albumin and 11.7 µg transferrin per 106 cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AFAF per mg cell protien. Hence, Ha-ras
EJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.Abbreviations
BWE
basal Williams' medium E
- FBS
fetal bovine serum
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F10 or F12
basal Ham's F10 or F12 medium
- Ha-ras
EJ
EJ allele of the human cellular ras oncogen of Harvey
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hGH
human growth hormone
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hsp
heat shock protein gene
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LE-p
liposome encapsulated plasmid
- N-OH-2-AFAF
N-hydroxy-2-acetylaminofluorene
- RLECC
rat liver epithelial cell
- SF
serum-free
- SS
serum-supplemented
- UGG
serum substitute UGltroser G®
- 1-OH-, 3-OH-2-AFAFF
1-hydroxy-, 3-hydroxy-2-acetylaminofluorene
- 2-AFAF
2-acetylaminofluorene
- 2-AFF
2-aminofluorene |
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Keywords: | Hepatocyte Ha-ras
EJ cell transformation liver cell line liposome encapsulated plasmid carcinogen metabolic activation 2-AFAF bile acids albumin transferrin hGH |
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