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背景:在神经细胞培养中实验性缺氧缺糖在一定程度上模拟缺血性卒中,对于研究缺血性神经元损伤的进程和病理生理学机制有非常重要的用处。目的:在神经元培养时制作实验性缺氧缺糖模型。设计、时间及地点:分组对照观察,实验于2007-01/2008-03在北京大学第三医院中心实验室完成。材料:17～19d胎龄的Wistar大鼠。方法:细胞培养取17～19d胎龄的Wistar大鼠的皮质神经元做原代细胞培养,并且去掉污染的非神经原细胞。缺氧缺糖的诱导分为3组:实验组将第7天的皮质神经元置于无糖平衡盐溶液和2%去氧酶中,在37℃的潮湿保温箱中培育。空白对照组培养基为含20mmol/L葡萄糖的无去氧酶平衡盐溶液。假性实验组培养基为含20mmol/L葡萄糖和失活的去氧酶平衡盐溶液。主要观察指标:以血气分析进行氧浓度的测定;以相差显微镜观察实验组培养细胞神经元死亡状况;以用乳酸脱氢酶检测盒检测乳酸脱氢酶活性;以锥虫蓝染色观察缺氧缺糖对神经元存活力的影响。结果:氧浓度测定显示在加入去氧酶后培养基迅速产生缺氧状态;乳酸脱氢酶检测显示在用去氧酶和无糖平衡盐处理后,培养基中乳酸脱氢酶释放显著增加;锥虫蓝染色和相差显微镜检查显示经去氧酶和无糖平衡盐处理后实验组的细胞活力明显下降,大部分神经元在6h死亡。结论:实验结果显示去氧酶与无糖平衡盐液可联合用于神经元培养时产生缺氧缺糖状态,其在体外模拟脑缺血的相关研究中有重要作用。
BACKGROUND: Experimental oxygen-glucose-deficient hypoglycemia in neuronal cultures mimics ischemic stroke to some extent and plays an important role in studying the process and pathophysiological mechanisms of ischemic neuronal injury. OBJECTIVE: To create an experimental model of hypoxia-deficient glucose during neuron culture. DESIGN, TIME AND SETTING: The grouping control study was performed at the Central Laboratory of Peking University Third Hospital from January 2007 to March 2008. MATERIALS: Wistar rats 17 to 19 days old. METHODS: Cell culture Cortical neurons from Wistar rats 17 to 19 days old were cultured in primary culture and contaminated non-neuronal cells were removed. The induction of oxygen and glucose deprivation was divided into three groups: In the experimental group, the cortical neurons on day 7 were placed in sugar-free balanced salt solution and 2% deoxygenase and incubated in a humid incubator at 37 ° C. Blank control group medium containing 20mmol / L glucose without deoxygenase balanced salt solution. Pseudo experimental group medium containing 20mmol / L glucose and inactivated deoxygenase balanced salt solution. MAIN OUTCOME MEASURES: Oxygen concentration was determined by gas analysis; neuron death in cultured cells was observed by phase contrast microscopy; lactate dehydrogenase activity was detected by lactate dehydrogenase detection kit; Effect of sugar on neuronal viability. Results: The oxygen concentration assay showed that the medium rapidly produced hypoxia after addition of deoxygenase; lactate dehydrogenase assay showed that lactate dehydrogenase release was significantly increased in the medium after treatment with deoxygenase and sugar-free balanced salt; Trypan blue staining and phase contrast microscopy showed that the viability of the experimental group was significantly decreased after treated with deoxygenase and sugar-free balanced salt, most of the neurons died at 6h. CONCLUSION: The results show that the combination of deoxygenase and sugar-free balanced salt solution can be used in neuronal culture to produce oxygen and glucose deprivation, which play an important role in the related research of simulated cerebral ischemia in vitro.