Paul D, Höhne M, Pinkert C, Piasecki A, Ummelmann E, Brinster R L
Department of Cell Biology, Fraunhofer-Institute for Toxicology, Hannover, Federal Republic of Germany.
Exp Cell Res. 1988 Apr;175(2):354-62. doi: 10.1016/0014-4827(88)90199-1.
Hepatocytes of transgenic mouse fetuses harboring SV40 virus transforming gene sequences in the SV delta e-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 alpha-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 formation in vivo.
在小鼠金属硫蛋白（MT-I）增强子驱动的SVδe-MGH融合基因构建体202中携带SV40病毒转化基因序列的转基因小鼠胎儿肝细胞，于妊娠第19天进行培养，并建立了一个表达白蛋白和甲胎蛋白（AFP）mRNA的分化细胞系。肝细胞系FMH-202含有整合的SV40序列，表达SV40 T抗原基因，并表现出无限生长潜力，因为它已培养18个月，细胞活力和生长速率均无明显下降，这表明未出现危机期。永生化细胞在缺乏精氨酸、添加转铁蛋白和胰岛素的化学限定培养基中增殖，而在原代培养中，表皮生长因子（EGF）、胰岛素和转铁蛋白是胎儿或新生小鼠肝细胞增殖的必需条件。细胞在琼脂中不生长，在裸鼠中也不具有致瘤性。汇合单层细胞的低饱和密度显示无过度生长，以及在缺乏胰岛素时生长停滞，随后在胰岛素作用下诱导DNA合成并恢复细胞生长，进一步证明了它们的永生化、非恶性表型。因此，看来永生化的表达SV40 T抗原的肝细胞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。
