IMMORTALIZED DIFFERENTIATED HEPATOCYTE LINES DERIVED FROM TRANSGENIC MICE HARBORING SV40 T-ANTIGEN GENES
IMMORTALIZED DIFFERENTIATED HEPATOCYTE LINES DERIVED FROM TRANSGENIC MICE HARBORING SV40 T-ANTIGEN GENES
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DOI：
10.1016/0014-4827(88)90199-1
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发表时间：
1988-04-01
影响因子：
3.7
通讯作者：
BRINSTER, RL
中科院分区：
文献类型：
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作者：
PAUL, D;HOHNE, M;BRINSTER, RL
转基因小鼠胎儿的肝细胞在由小鼠金属硫蛋白(MT-I)增强子驱动的SVΔe-MGH融合基因构建体202中含有SV40病毒转化基因序列[R. D. Palmiter、H. Y. Chen、A. Messing 和 R. L. Brinster (1985)Nature (London)316, 457–460] 在妊娠第 19 天进行培养，并建立为表达白蛋白和甲胎蛋白 (AFP) mRNA 的分化系。肝细胞系FMH-202含有整合的SV40序列，表达SV40 T抗原基因，并表现出无限的生长潜力，因为它已经培养了18个月，细胞活力或生长速度没有明显下降，这可能表明危机期的发生。永生化细胞在缺乏精氨酸、转铁蛋白和胰岛素的化学成分确定的培养基中增殖，而EGF、胰岛素和转铁蛋白是原代培养物中胎儿或新生小鼠肝细胞增殖的必需要求。细胞不在琼脂中生长，并且在裸鼠中不致瘤。他们永垂不朽。非恶性表型进一步记录为汇合单层细胞的低饱和密度，未显示过度生长，以及在没有胰岛素的情况下生长停滞，随后诱导 DNA 合成并响应胰岛素恢复细胞生长。因此，表达永生化SV40 T抗原的肝细胞似乎存在于转基因小鼠的肝脏中。然而，在肝脏发育的后期，T抗原的转化活性变得明显并导致体内肝细胞癌的形成。
Hepatocytes of transgenic mouse fetuses harboring SV40 virus transforming gene sequences in the SVΔe-MGH fusion gene construct 202 driven by the mouse metallothionein (MT-I) enhancer [R. D. Palmiter, H. Y. Chen, A. Messing, and R. L. Brinster (1985)Nature (London)316, 457–460] were cultured at Day 19 of gestation and established as a differentiated line expressing albumin and α-fetoprotein (AFP) mRNAs. Hepatocyte line FMH-202 contains integrated SV40 sequences, expresses SV40 T-antigen genes, and exhibits unlimited growth potential because it has been cultured 18 months without apparent decrease in cell viability or in growth rate that could suggest the occurrence of a crisis period. Immortalized cells multiply in chemically defined medium deficient in arginine with transferrin plus insulin, whereas EGF, insulin, and transferrin are obligatory requirements for fetal or newborn mouse hepatocyte multiplication in primary cultures. Cells did not grow in agar and were not tumorigenic in nude mice. Their immortalized. nonmalignant phenotype was further documented by low saturation densities of confluent monolayers showing no overgrowth, and by growth arrest in the absence of insulin with subsequent induction of DNA synthesis and resumption of cell growth in response to insulin. Thus, it appears that immortalized SV40 T-antigen-expressing hepatocytes are present in the liver of the transgenic mice. However, at later points in liver development the transforming activity of T-antigen becomes apparent and leads to hepatocellular carcinoma formationin vivo.