Orloff S, Mukherjee A B, Butler J D, Foley B, Schulman J D
In Vitro. 1980 Aug;16(8):655-60. doi: 10.1007/BF02619194.
Extracellular cysteine concentrations between 0.5 and 2.5 mM resulted in death of normal but not cystinotic cells grown in Eagle's minimal essential medium containing supplemental fetal bovine serum and antibiotics. Differential cell survival was determined by viable cell counting using Trypan Blue dye exclusion. In cocultivation experiments of [3H]thymidine-labelled cystinotic fibroblasts with nonradioactive normal fibroblasts, autoradiography confirmed the selective survival of cystinotic cells in medium containing 1 mM cysteine. At this concentration of 1 mM cysteine, intracellular cystine content increased slightly in surviving normal cells but not in cystinotic cells, which normally contain a high level of intracellular cystine. This comparative resistance of cystinotic fibroblasts to elevated extracellular cysteine concentrations forms the basis for an in vitro selective system for these mutant human cells. Further exploration of this resistance phenomenon may well expand the understanding of the molecular defect in cystinotic cells.
在含有补充胎牛血清和抗生素的伊格尔氏最低限度基本培养基中培养时，细胞外半胱氨酸浓度在0.5至2.5 mM之间会导致正常细胞死亡，但不会导致胱氨酸病细胞死亡。通过使用台盼蓝染料排除法进行活细胞计数来确定细胞的差异存活情况。在用[3H]胸腺嘧啶核苷标记的胱氨酸病成纤维细胞与无放射性的正常成纤维细胞进行共培养实验中，放射自显影证实了胱氨酸病细胞在含有1 mM半胱氨酸的培养基中的选择性存活。在1 mM半胱氨酸的这个浓度下，存活的正常细胞内胱氨酸含量略有增加，但胱氨酸病细胞内的胱氨酸含量没有增加，而胱氨酸病细胞通常含有高水平的细胞内胱氨酸。胱氨酸病成纤维细胞对升高的细胞外半胱氨酸浓度的这种相对抗性构成了这些突变人类细胞体外选择系统的基础。对这种抗性现象的进一步探索很可能会扩展对胱氨酸病细胞分子缺陷的理解。
