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目前对缺血性神经元损伤机制的研究主要聚焦在究竟是调亡或是坏死机制导致神经元损伤的问题上,因为不同的机制涉及到不同治疗方法的探讨.本研究用新生鼠(出生<24h)海马神经元分散培养7～10d后,建立低氧缺血模型,即分别加入1mM Glutamate(高谷氨酸组)10min;100μM NaCN(低氧组)10min;100μM NaCN+3.5nMIodoacete(缺血组)10min;200μDMEM-without glucose(缺葡萄糖组)2h和100%N(缺氧组)1h.PBS漂洗细胞后,滴加50μAnnekin-V免疫荧光标记液(标记液含:20μAnnexin-V-flous,20μl Propidium iodide,1000 μl Propidium iodide,1000 μl Hepes buffer),37℃乱砍滥伐孵育15min, Olympus荧光显微镜下随机选择三个视野计数FTTC单标细胞(凋亡细胞)和fTTC+PI双标细胞(坏死细胞).结果显示低氧缺血诱导的凋亡神经元胞体和突起有强绿色荧光标记,但细胞核无PI着色.坏死神经元除胞膜有绿色荧光外,胞核显示红色强PI染色,且坏死神经元多成群聚集,而凋亡神经元则分散分布.统计结果表明缺血组和缺葡萄糖组的凋亡率最高(前者为58.5 ±9.5;后者为49.5±8.6).谷氨酸组、低氧组、缺氧组凋亡率较低,且组间无显著性差异.正常细胞膜磷脂双层结构是不对称的.细胞凋亡时,由于大量增加的胞浆Ca~(2+)对脂质爬行酶和氨基磷脂转位酶活
At present, the research on the mechanism of ischemic neuronal damage mainly focuses on whether neuronal damage is caused by apoptosis or necrosis, because different mechanisms involve different therapeutic approaches.In this study, 24h) hippocampal neurons were cultured for 7 ~ 10d, hypoxia-ischemia model was established by adding 1mM Glutamate 10min, 100μM NaCN 10min, 100μM NaCN + 3.5nMI odoacete ) For 10 min, 200 μM DMEM without glucose for 2 h and 100% N for 1 h. After the cells were washed by PBS, 50 μAnnekin-V immunofluorescent labeling solution (20μAnexnex-V-flous) Propidium iodide, 1000 μl of Propidium iodide, 1000 μl of Hepes buffer) were added and de-cut at 37 ℃ for 15 min. Three FTs were selected under Olympus fluorescence microscope to count FTTC single-labeled cells (apoptosis cells) and fTTC + PI double-labeled cells Necrotic cells) .Results showed that hypoxia-ischemia-induced apoptotic neuronal somatic cells and protuberances had strong green fluorescence labeling, but no PI staining of nuclei.Nuclei showed red PI staining in addition to green fluorescence in the necrotic neurons, And more neurons into necrosis While the apoptotic neurons were scattered.The statistical results showed that the apoptotic rates of the ischemic group and the dextrose group were the highest (the former was 58.5 ± 9.5 and the latter was 49.5 ± 8.6) The apoptotic rate of hypoxia group was lower, and there was no significant difference between the two groups.The asymmetry of double membrane structure of phospholipid in normal cell membrane was observed.When apoptosis was increased, a large amount of cytosolic Ca ~ (2+) And aminophospholipid translocase activity