胆红素诱导原代培养神经细胞内Ca2+含量的变化及MgSO4的拮抗作用

Bilirubin-induced Increase in Intracellular Ca2+ in Primary Cultured Neurons and the Reversible Effect of MgSO4

摘要:

目的 探讨胆红素诱导原代培养神经细胞内Ca2 + 含量的变化 ,以及MgSO4 的拮抗作用。方法 采用胎鼠大脑皮层细胞作原代神经细胞培养 ,观察 10 0 μmol/L胆红素对不同成熟程度神经细胞内Ca2 + 含量的影响。原代培养神经细胞经Fura- 2 /AM负载 ,荧光图像分析测定细胞内Ca2 + 含量。结果 未成熟神经细胞 (培养2天 )暴露于 10 0 μmol/L胆红素 4h ,细胞内Ca2 + 从 75 .3nmol增加到 32 0 .8nmol;继续暴露 4h ,细胞内Ca2 + 增加到 40 0 .7nmol;去除胆红素暴露 ,细胞内Ca2 + 不能逆转。成熟神经细胞 (培养 8天 )暴露于 10 0 μmol/L胆红素 4h ,细胞内Ca2 + 从 70 .6nmol增加到 15 0 .8nmol;继续暴露 4h ,细胞内Ca2 + 不再增加 ;去除胆红素暴露 ,细胞内Ca2 + 浓度可显著降低。培养液中Mg2 + 加至 2mmol/L可减轻胆红素诱导的神经细胞内Ca2 + 含量变化。结论 胆红素可诱导原代培养的、未成熟的神经细胞Ca2 + 超载 ,MgSO4 可拮抗胆红素诱导的神经细胞Ca2 + 超载。

Abstract:

Objective To explore the effect of bilirubin on intracellular Ca 2+ of cerebral cortex neurons and the therapeutic effect of MgSO 4. Methods Cerebral cortical neurons were cultured from the embryonic rat brain. The free cytosolic Ca 2+ concentration ([Ca 2+ ]i) of the cultured neurons was determined with a fluorescent Ca 2+ chelator (Fura-2) after exposure of the neurons to different concentrations of bilirubin. Results Immature neurons (neurons cultured for only 2 days) responded within 4 hours to 100 mmol bilirubin by an increase in [Ca 2+ ]i from 75.3 nmol to 320.8 nmol, an increase that during another 4 hours of exposure reached to 400.7 nmol. This increase in [Ca 2+ ]i could not be reversed by the removal of bilirubin. In contrast to these findings, mature neurons (8 days in culture) exhibited only a small increase (from 70.6 nmol to 150.8 nmol) in [Ca 2+ ]i within 4 hours after the exposure to 100 mmol bilirubin, and this rapid increase in [Ca 2+ ]i tended to decline on the following prolonged 4 hour exposure to bilirubin. Moreover, after the removal of bilirubin, the increase in [Ca 2+ ]i was partially reversible. The N-methyl-D-aspartate (NMDA) receptor antagonists MgSO 4 could significantly block the response. Conclusions Bilirubin can induce an increase in intracellular Ca 2+ in primary cultured cerebral cortex neurons and the change can be partially reversed by NMDA receptor antagonists MgSO 4.