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目的探讨模拟缺血再灌注引起神经元凋亡的途径和褪黑素(m elaton in,MT)抗凋亡的作用机理。方法建立原代培养大鼠小脑颗粒细胞的体外模拟缺血(Oxygen G lucose Deprivation,OGD)再灌注模型,测定培养液LDH活性和细胞DNA琼脂糖凝胶电泳;利用Rhodam ine123和激光共聚焦显微镜观察线粒体膜电位的变化;ELISA检测细胞浆中细胞色素C水平;用同样指标观察MT对其损伤的保护作用。结果在体外模拟缺血再灌注模型中培养液LDH活性增加(P<0.05),琼脂糖凝胶电泳DNA出现梯状条带,线粒体膜电位明显降低,细胞浆中细胞色素C含量增加(P<0.05),MT对上述现象有明显的抑制作用。结论(1)缺血再灌注引起的神经元凋亡机理之一是通过线粒体凋亡途径;(2)MT可通过阻止线粒体凋亡途径而保护模拟缺血再灌注诱导小脑颗粒细胞的凋亡。
Objective To investigate the pathways that mimic ischemia-reperfusion-induced neuronal apoptosis and the mechanism of anti-apoptotic effect of m elaton in (MT). Methods Primary cultured rat cerebellar granule cells were established by OGD reperfusion. The LDH activity and DNA agarose gel electrophoresis were measured. Mitochondrial membrane potential changes; ELISA cytosolic cytochrome C levels; with the same indicators to observe the MT of its protective effect. Results The LDH activity increased (P <0.05) in the model of ischemia-reperfusion injury in vitro. The ladder-like DNA appeared on the agarose gel electrophoresis DNA, the mitochondrial membrane potential was significantly decreased and the content of cytochrome C in cytoplasm increased (P < 0.05), MT significantly inhibited the above phenomenon. Conclusion (1) One of the mechanisms of neuronal apoptosis induced by ischemia-reperfusion is through the mitochondrial apoptosis pathway. (2) MT can protect the apoptosis of cerebellar granule cells induced by simulated ischemia-reperfusion by blocking the mitochondrial apoptosis pathway.