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观察磷酸化 C- JUN与谷氨酸诱导的小脑颗粒神经元凋亡的关系。在培养的小脑颗粒神经元建立谷氨酸凋亡模型 ;采用 MTT法分析细胞存活率 ,相差显微镜观察形态学 ,DNA凝胶电泳法分析细胞凋亡和原位细胞荧光免疫组织化学法检测磷酸化 C- JUN。结果显示 ,谷氨酸诱导大鼠小脑颗粒神经元细胞体积缩小 ,突触断裂、消失 ,DNA电泳呈典型的“梯状”条带 ;谷氨酸处理 2 4 h后细胞存活率为 2 8.6%± 5.2 %。神经元在谷氨酸处理 5,30 min及 1 ,2 ,4,8,1 6和 2 4 h后均未检测到有磷酸化 C- JUN阳性细胞 ,与去极化组 ( 2 5mmol/L KCl)相同。而复极化组 ( 5mmol/L KCl)则在 30 min检测到大量的磷酸化 C- JUN阳性细胞 ,4h荧光最强并持续。处理 4h后 ,40 0倍荧光显微镜下 ,复极化组、去极化组和谷氨酸组的磷酸化 C- JUN阳性细胞数分别为 1 2 4± 1 7,8± 3,5± 3。上述结果提示 ,谷氨酸诱导小脑颗粒神经元凋亡 ,磷酸化 C- JUN不参与谷氨酸诱导的大鼠小脑颗粒神经元凋亡。
To observe the relationship between phosphorylated C-Jun and glutamate-induced cerebellar granule neuronal apoptosis. Glutamate apoptosis model was established in cultured cerebellar granule neurons; cell viability was analyzed by MTT method, morphology was observed by phase contrast microscopy, apoptosis was analyzed by DNA gel electrophoresis and fluorescence in situ was detected by fluorescence immunohistochemistry C- JUN. The results showed that the glutamate-induced rat cerebellar granule neurons decreased in size and synapse ruptured and disappeared, and DNA ladder showed a typical “ladder-like” band. After 24 h of glutamate treatment, the cell survival rate was 22.66% ± 5.2%. Neurons were not detected phosphorylated C-JUN positive cells at 5,30 min and 1,2, 4, 8, 1, 6 and 24 h after glutamate treatment. Compared with the depolarization group (25 mmol / L KCl) the same. In the repolarization group (5 mmol / L KCl), a large amount of phosphorylated C-JUN positive cells were detected at 30 min, and the fluorescence was the strongest at 4 h and continued. After 4 hours of treatment, the number of phosphorylated C-JUN positive cells in the repolarization group, depolarization group and glutamic acid group were respectively 124 ± 1 7, 8 ± 3, 5 ± 3 . The above results suggest that glutamate induced apoptosis of cerebellar granule neurons and phosphorylated C-JUN did not participate in the glutamate-induced apoptosis of rat cerebellar granule neurons.