制备神经细胞实验性缺氧缺糖模型的简易方法

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:17-19 d胎龄的Wistar大鼠.方法:细胞培养取17～19 d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甲衡盐溶液和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20 mmol/L葡萄糖的无去氧酶平衡盐溶液.假性实验组培养基为含20 mmol/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6 h死亡.结论:实验结果显示去氧酶与无精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.

An easy and effective way to produce experimental oxygen-glucose deprivation in cultured neurons

BACKGROUND: Oxygen-glucose depdvation (OGD) in cultured neurons simulates stroke to a certain degree and plays an important role in studying processing and pathophysiological mechanism of ischemic neuronal injury.OBJECTIVE: To produce experimental OGD models in cultured neurons.DESIGN, TIME AND SETTING: A grouping controlled study was performed at the Center Laboratory of Third Hospital, Peking University from January 2007 to March 2008.MATERIALS: Fetal Wistar rats with gestational age of 17-19 days were collected in this study.METHODS: Primary cultures of cortical neurons that were derived from fetal Wistar rats with gestational age of 17-19 days were performed to remove pollutional non-neuronal cells. OGD was produced by incubation with non-glucose balanced salt solution and 2% Oxyrasa in 7-day cultured cortical neuron cultures. Cell cultures were kept in a humidified 37 ℃ incubator. In the control group, cell culture medium was replaced with balanced salt solution containing 20 mmol/L glucose. In the sham operation group,balanced salt solution containing 20 mmol/L glucose and Oxyrasawere used to replace the medium.MAIN OUTCOME MEASURES: Oxygen concentration in the culture medium was measured with blood gas analysis; neuronal death in the experimental group was observed under phase contrast microscope; lactate dehydrogenase activity was detected with lactate dehydrogenasa assay; effect of oxygen-glucose deprivation on neuronal viability was observed with trypan blue staining.RESULTS: Measurement of oxygen concentration showed that hypoxia could be quickly achieved shortly after the addition of Oxyrase; lactate dehydrogenase assay revealed that after treatments of neuron cultures with Oxyrase and non-glucose balanced salt solution, lactate dehydrogenase release increased significantly with the treatment time; trypan blue staining and phase contrast microscope showed that cell viability decreased after treatments of Oxyrase and non-glucose balanced salt solution, and most neurons died 6 hours after OGD.CONCLUSION: These results show that Oxyrase, together with non-glucose balanced salt solution, can be conveniently used to produce OGD condition in cultured neuronal cells which is greatly useful in the study of simulating cerebral ischemia in vitro.