QUANTITATIVE ASSAY FOR ALBUMIN-PRODUCING LIVER-CELLS AFTER SIMIAN VIRUS-40 TRANSFORMATION OF RAT HEPATOCYTES MAINTAINED IN CHEMICALLY DEFINED MEDIUM
QUANTITATIVE ASSAY FOR ALBUMIN-PRODUCING LIVER-CELLS AFTER SIMIAN VIRUS-40 TRANSFORMATION OF RAT HEPATOCYTES MAINTAINED IN CHEMICALLY DEFINED MEDIUM
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DOI：
10.1073/pnas.81.20.6378
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
GEORGOFF, I
中科院分区：
文献类型：
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作者：
ISOM, HC;GEORGOFF, I
在化学成分确定的培养基中检测了SV 40对大鼠肝细胞的转化。当接种在胶原包被的平板上的肝细胞被SV 40感染时，复制细胞的菌落早在感染后40天就出现了;在对照培养物中没有菌落出现。来自85%转化培养物的培养基含有白蛋白。当胶原蛋白被消除和heptocytes被铺板在Primaria塑料细胞培养皿，转化发生;培养基中86%的转化培养物含有白蛋白，但分泌的最大白蛋白水平，每个文化只有62%的胶原蛋白涂层的植物上产生的文化。建立了转化的定量测定方法。每个肝细胞感染2-50个噬斑形成单位的病毒后，转化呈线性，在2个平板表面上的转化频率相同。免疫覆盖技术使得有可能识别、纯化和确定白蛋白产生细胞的形态。当培养基中的精氨酸被鸟氨酸取代时，转化频率显著降低，而产生白蛋白的菌落百分比从30%增加到100%。定义了一种用于定量正常肝细胞群体转化以及用于鉴定和计数产生白蛋白的上皮肝细胞转化体的测定法。
Transformation of rat hepatocytes by SV40 in chemically defined medium was examined. When hepatocytes plated on collagen-coated plates were infected with SV40, colonies of replicating cells appeared as early as 40 days after infection; no colonies appeared in control cultures. Medium from 85% of the transformed cultures contained albumin. When collagen was eliminated and heptocytes were plated on Primaria plastic cell culture dishes, transformation occurred; medium from 86% of the transformed cultures contained albumin but the maximum albumin level secreted per culture was only 62% of that produced by cultures on collagen-coated plants. Quantitative assays for transformation were established. Transformation was linear after infection with 2-50 plaque-forming units of virus per hepatocyte, and the transformation frequency was the same on the 2 plating surfaces. An immuno-overlay technique made it possible to identify, purify and determine the morphology of the albumin-producing cells. When ornithine was substituted for arginine in the medium, the transformation frequency decreased markedly while the percentage of colonies producing albumin increased from 30 to 100%. An assay for quantifying transformation of a normal hepatocyte population and for identifying and enumerating epithelial liver cell transformants that produce albumin was defined.