Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts.
Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts.
复制标题
细胞培养条件对人生殖器皮肤成纤维细胞芳香酶活性的影响。
DOI：
10.1007/bf02623664
复制
发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Berkovitz,GD
中科院分区：
文献类型：
--
作者：
Bisat,T;Brown,TR;Migeon,CJ;Berkovitz,GD
由于测定培养的人生殖器皮肤成纤维细胞中芳香化酶活性已被提议作为研究男性雌激素产生的一种手段，我们研究了培养条件对芳香化酶活性的影响。期酶活性在前14天缓慢上升，但随后迅速上升至第28天的10倍高的平台。芳香化酶活性的上升是相似的，无论是蛋白质或DNA含量的活性归一化。当以1× 106或0.25× 106细胞/板的常规密度接种细胞时，低密度接种细胞的芳香化酶活性在前2 wk始终较低，但此后两组的酶活性水平趋于一致。在以较低密度接种的细胞中，在前2周观察到的较低活性与较低的Vmax相关。将一种密度的细胞与另一种密度的细胞的条件培养基预孵育，两组中的相对活性水平没有改变。相比之下，双氢睾酮（DHT）受体结合和5α-还原酶活性在所有时间点是相似的，尽管不同的平板密度。在额外的实验中，每天更换培养基，而不是每3天，并在第7天测定芳香酶活性。每天补料的细胞DNA和蛋白质含量是每3天补料细胞的2倍。相比之下，芳香化酶活性下降到后一组的30%。两组的DHT和地塞米松受体结合率和5α-还原酶活性相似。总之，接种密度、培养密度和培养基更换频率等因素显著影响培养的人生殖器皮肤成纤维细胞中芳香化酶活性。因此，从培养细胞中获得的芳香酶活性数据与生理或病理状态的关系的解释应适当谨慎。
Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying estrogen production in men, we investigated the influence of culture conditions on aromatase activity.Genital skin fibroblasts were seeded onto culture plates at a density of 1×106cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized for protein or for DNA content. When cells were seeded at the usual density of 1×106or at 0.25×106cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed in the first 2 wk was associated with a lower Vmax. Preincubation of cells plated at one density with conditoned medium from cells plated at the other density did not change the relatve levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5α-reductase activity were similar at all time points, despite differences in plating density.In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity declined to 30% of the in the latter group. DHT and dexamethasone receptor binding and 5α-reductase activity were similar in the two groups.In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution.