【摘要】 目的 :通过应用共聚焦激光扫描显微镜技术 (confocallaserscanningmicroscope ,CLSM)观察脑缺血后谷氨酸载体GLASTmRNA和EAAT1蛋白表达的细胞定位 ,探讨CLSM技术中三维重建和三维显示在观察基因和蛋白在神经细胞上空间定位的应用。方法 :对脑缺血后采用原位杂交和免疫组织化学相结合的荧光双标技术标记的脑片进行双通道断层扫描以及三维数据重组。结果 :脑缺血后 ,荧光免疫组化双标显示大脑皮质缺血半暗区内的谷氨酸载体EAAT1蛋白与星形胶质细胞和神经元均有共表达 ;原位杂交结合免疫组化的荧光双标显示缺血周边区谷氨酸载体GLASTmRNA与星形胶质细胞和神经元有明显的共表达。结论 :共聚焦激光扫描显微镜观察脑缺血后谷氨酸载体GLASTmRNA和EAAT1蛋白在神经胶质细胞和神经元上的空间定位 ,为进一步研究脑缺血后谷氨酸载体系统作用机制提供了更为准确的形态依据。更多还原

【Abstract】 Aim:Series and 3D image of confocal laser scanning microscopy(CLSM) combined with in situ hybridization and immuno-fluorescent double labeling techniques were used to study cellular localization of glutamate transporter GLAST mRNA and EAAT 1 protein expression in glial and neuronal cells of adult rat at 72 h following cerebral ischemia. Methods:After double labeling,expression of GLAST mRNA and EAAT 1 were detected in glial and neuronal cells of brain after ischemia injury by two channel scanning and 3D image of CLSM.Results:Double staining showed that glutamate transporter GLAST mRNA and EAAT 1 protein co-expressed with in some glial cells and neurons in the area penumbra of cerebral cortex.Conclusion:The results suggest that increase of GLAST mRNA and EAAT 1 protein expression in the area penumbra at 72 h postischemia possibly represent a protective mechanism for increasing extracelluar glutamate uptake and limiting glutamate neurotoxicity. Confocal laser scanning microscopy offered a real three-dimensional image for observing cellular localization of glutamate transporter GLAST mRNA and EAAT 1 protein expression in glial and neuronal cells of brain after ischemia injury.更多还原