Studies on the alteration of calcium ions in osteoblastic cell line using a fluorescence dye

在小鼠成骨细胞系MC 3 T3-E1细胞中，用荧光分光光度计和荧光显微镜测量系统，使用细胞内Ca^<2+>离子荧光试剂，研究Ca^<2 +>离子的内流和细胞内变化。在人表皮样癌细胞系A431中观察到对表皮生长因子（EGF）有反应的Ca^2+离子的一次通过内流，但在成骨细胞系MC 3 T3-E1中没有观察到。然而，在两种细胞系中均检测到细胞内游离Ca^2+浓度对胎牛血清（FCS）的反应性增加。以一次传代的方式，FCS处理后的成骨细胞MC 3 T3-E1出现了对EGF敏感的钙离子内流，提示EGF与FCS中的任何一种因子协同作用，均可诱导MC 3 T3-E1细胞内少量Ca^2+内流，提示EGF-E1可能通过一种非常微弱的信号转导途径，EGF受体系统在MC 3 T3-E1细胞生长初期起协同作用。另一方面，我们还发现成骨细胞MC 3 T3-E1中表达神经营养素-3（NT-3：神经生长因子之一）及其受体（TRKC），并且成骨细胞中发生对NT-3应答的Ca^2+内流。此外，用荧光显微测量系统对MC 3 T3-E1细胞内Ca^2+浓度变化进行的光作图数据表明，MC 3 T3-E1细胞内Ca ^2+浓度高于A431细胞。这表明成骨细胞特有的细胞内Ca^2+积累机制在成骨细胞系统中以不同于其他细胞如表皮细胞的方式起作用。

In mouse osteoblastic clone MC3T3-E1 cells, influx and intracellular alteration of Ca^<2+>ions were investigated with the fluorescence-spectrophotometer and fluorrescence-microscopic measurement system using a fluorescence reagent for intracellular Ca^<2+>ions. One passing influx of Ca^<2+>ions responsive to epidermal growth factor (EGF) was observed in human epidermoid carcinoma cell line : A431, but not in the osteoblastic cell line : MC3T3-E1 with both the fluorescence measurements. However, the increase of intracellular concentration of free Ca^<2+>ions responsive to fetal calf serum (FCS) was detected in both cell lines. With the manner of one passage, futhermore, influx of Ca^<2+>ions responsive to EGF appeared in the osteblastic MC3T3-E1 cells after the FCS-treatment, suggesting that a slight influx of Ca^<2+>ions into the MC3T3-E1 cells is induced with the synergistic function of EGF and any factor (s) in FCS.These results indicate that a very silght signal transduing due to the EGF-EGF receptor system is functioning by the synergistic effect during the initial growth phase of MC3T3-E1 cells. On the other hand, we also found that neurotrophin-3 (NT-3 : one of nerve growth factors) and its receptor (TRKC) is expressed in the osteoblastic MC3T3-E1 cells and the influx of Ca^<2+>ions responsive to NT-3 occurs in the osteoblastic cells. This implies that the signal transdicing sytem due to the interaction between NT-3 and TRKC is acting during the initial growth of MC3T3-E1.Moreover, photomapping data on the alteration of intracellular Ca^<2+>concentration detected with a fluorescence-microscopic measurement system indicated that the intracellular Ca^<2+>accumulation in MC3T3-E1 cells is higher than that in A431 cells. This suggests that an osteoblast-specific mechanism of intracellular Ca^<2+> accumulation plays in the osteoblastic cell system with the different manner from other cells such as epidermal cells.