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目的探讨脑缺血后细胞凋亡发生的可能机制以及神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、p38丝裂原活化蛋白激酶(mitogen activated proteinkinasep38,p38MAPK)和半光氨酸蛋白酶-3(caspase-3)在脑缺血后神经细胞凋亡中的共同作用机制。方法采用线栓法闭塞大鼠大脑中动脉(middle cerebral artery occlusion,MACO)建立脑缺血SD大鼠模型,应用透射电镜观察脑缺血对脑组织超微结构的影响,流式细胞仪方法(FCM)分别定量检测细胞凋亡率,半定量RT-PCR检测nNOS、iNOS,p38MAPK和Caspase-3mRNA表达水平。结果透视电镜下脑缺血6h出现核固缩,缺血12h出现细胞核分裂,缺血24h出现凋亡小体;FCM检测细胞凋亡百分率随着缺血时间延长而增加,缺血72h达到高峰,约70.37%;RT-PCR产物的琼脂糖凝胶电泳显示nNOS、iNOS、p38MAPK和Caspase-3mRNA的特异性片段大小分别为501、342、250和342bp,但mRNA表达量不一致,nNOS mRNA主要在缺血早期表达,iNOS、p38MAPK和Caspase-3mRNA在缺血中晚期表达,并在缺血3~5d,后三种基因的表达量达到高峰。结论脑缺血区域发生典型的神经细胞凋亡现象,nNOS来源的NOS在缺血早期发挥神经毒性作用,iNOS来源的NOS在缺血晚期发挥神经毒性作用;NOS,p38MAPK和Caspase-3三种基因的相互关系可能构成介导缺血神经细胞凋亡的通路之一。
Objective To investigate the possible mechanism of apoptosis after cerebral ischemia and the effects of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), p38 mitosis Mechanisms of mitogen activated protein kinase (p38MAPK) and caspase-3 in neuronal apoptosis after cerebral ischemia. Methods Middle cerebral artery occlusion (MACO) was used to establish the model of cerebral ischemia in rats. The effects of cerebral ischemia on the ultrastructure of cerebral tissue were observed by transmission electron microscopy. Flow cytometry FCM) were used to detect the apoptosis rate. The expression of nNOS, iNOS, p38MAPK and Caspase-3 mRNA were detected by semi-quantitative RT-PCR. Results Nuclear pyknosis was observed at 6h under electron microscope. Nuclear was observed at 12h after ischemia. Apoptotic bodies were observed at 24h after ischemia. The percentage of apoptotic cells increased with the prolongation of ischemia and peaked at 72h after ischemia. About 70.37%. The specific fragment sizes of nNOS, iNOS, p38MAPK and Caspase-3 mRNA of RT-PCR products were 501, 342, 250 and 342bp, respectively. However, the mRNA expression was not consistent and nNOS mRNA was mainly in the absence Early expression of blood, iNOS, p38MAPK and Caspase-3 mRNA expression in the late ischemia, and 3 to 5 days after ischemia, the three genes reached a peak expression level. CONCLUSIONS: Typical neuronal apoptosis occurs in ischemic area. NOS derived from nNOS plays a neurotoxic role in the early stage of ischemia. NOS derived from iNOS exerts neurotoxicity in late stage of ischemia. NOS, p38MAPK and Caspase-3 May constitute one of the pathways mediating the apoptosis of ischemic neurons.