共聚焦激光扫描显微镜技术在研究大鼠海马神经元胞内钙超载中的应用

Application of Confocal Laser Scanning Microscopy on Study Calcium Overload in Rat Hippocampal Neurons

目的 观察抑制性氨基酸牛磺酸对兴奋性氨基酸谷氨酸所致胞内钙超载的影响。方法 选择Fluo-3／ AM对培养的海马神经元进行着色后,用共聚焦激光扫描显微镜(confocal laser scanning microscope,CLSM)实时 扫描,动态显示海马神经元二维图像,以计算机相应软件对胞内钙的荧光强度变化进行分析统计。结果 在 0.5 mmol／L谷氨酸作用下,胞内钙荧光强度迅速升高(P<0.001);0.5 mmol／L谷氨酸与3 mmol／L牛磺酸共同 作用于海马神经元时,胞内钙荧光强度明显降低(P<0.001);灌流液中去除牛磺酸后,胞内钙荧光强度上升(P <0.001)。结论 抑制性氨基酸牛磺酸可抑制由兴奋性氨基酸谷氨酸所致神经元的胞内钙超载;CLSM以其独 特的优势,在高清晰度地显示海马神经元形态的同时,也可动态地观察活细胞在不同环境下胞内钙的快速变化 以及定量分析。该技术在动态观察和二维图像的高分辨率上比传统测钙方法有更大的优越性。

Purpose To observe real-time changes of intracellular calcium arose from glutamate and taurine in cultured rat hippocampal neurons. Methods After loading Fluo-3/AM in cultured rat hippocampal neurons, two dimensional image of intracellular calcium and analysis of fluorescence intensity were achieved by Confocal laser scanning microscopy (CLSM).Results An immediate peak of fluorescent intensity of [Ca2+]i was detected as soon as 0.5mmol/L glutamate was applied (P<0.001). Fluorescent intensity of [Ca2+]i reduced obviously using 0.5 mmol/L glutamate and 3 mmol/L taurine simultaneously (P< 0.001). After the removal of taurine,the rise of fluorescence intensity demonstrated the increase of [Ca+]i (P<0.001). Conclusions The results indicated that taurine in moderate concentration may exert antiexcitotoxic and antihypoxic effect partially via its antagonism to [Ca2+]i overload.CLSM can offer twodimensional calcium image of highly resolving power and real-time changes of intracellular calcium.