大鼠海马神经元体外氧糖剥夺／复氧模型的构建

The experimental probe into the construction of oxygen glucose deprivation/reoxygenation model of hippocampal neurons of rat in vitro

目的建立体外培养的大鼠海马神经元氧糖剥夺（OGD）／复氧实验模型，并尝试确定该模型最合适的缺氧缺糖时间点。方法新生SD乳鼠海马神经元原代培养7d后，随机（随机数字法）分为OGD组和对照组。OGD组根据氧糖剥夺时间的不同又分为1h，2h，4h，6h，8h，10h亚组。OGD组细胞置于含0．5％氧气的三气培养箱，同时将培养液换成无糖Earle氏液，模拟体内脑缺血损伤。复氧复糖24h后观察对照组和OGD各组的神经元形态，测定MTF细胞光密度值（OD）和培养液LDH含量，流式细胞仪检测细胞凋亡率。所得数据采用SPSS16．0统计软件行单因素方差分析（Durmett—t检验）和Spearman等级相关分析。结果随着缺氧缺糖时间的延长，OGD各组神经元形态学损伤逐步加重，细胞OD值和存活率逐渐下降（rs=-0．961和rs=-0．966，P〈0．01），LDH值逐渐升高（rs=0．990，P〈0．01），细胞凋亡率明显增加，与对照组比较差异均有统计学意义（P〈0．05）。OGD6h时，细胞的凋亡率接近50％。结论成功建立了大鼠海马神经元体外氧糖剥夺／复氧模型，结合形态学改变和细胞凋亡率，建议将6h作为该模型合适的缺氧缺糖损伤时间。

Objective To establish the oxygen glucose deprivation （OGD）/reoxygenation experimental model of hippocampal neurons of rat in vitro, and to try to identify the length of time for producing optimum injury in this model. Method The primary hippocampal neurons of newborn Sprague-Dawley rats were cultured for 7 days and randomly （random number） divided into a control group and OGD groups. The OGD groups were assigned into 1 h, 2 h, 4 h, 6 h, 8 h and 10 h subgroups in accordance with different lengths of time for oxygen glucose deprivation. The neurons of OGD groups were placed into a tri-gas incubator containing 0.5% oxygen and the culture medium was substituted with the glucose-free Earle＇ s balanced salt solution, simulating cerebral ischemia injury in vivo. The morphology of neurons was observed after reoxygenation for 24 hours. The MTT assay was used to determine the rate of survived cells derived from the value of optical density （OD） of cells. The lactate dehydrogenase （LDH） content in culture medium was detected to evaluate the neuron injury. The apoptotic rate of neurons was measured by using flow cytometry. Dunnett-test and Spearman correlation analysis were used to analyze the data with SPSS version 16.0 soft ware package. Results The morphological damage of neurons in OGD groups aggravated gradually, optical density and cell survival rate decreased （rs = -0.961 and rs = -0.966, P 〈 0.01）, and the amount of LDH increased （rs = 0. 990, P 〈 0.01 ） with longer duration of exposure to oxygen glucose deprivation, and the rate of neuron apoptosis increased obviously which was significantly statistical difference in com parison with the control group （ P 〈 0.05）. Under the setting of oxygen glucose deprivation for 6 hours, the apoptosis rate of neurons approximated to 50%. Conclusions The oxygen glucose deprivation/reoxygenation model of rat＇s hippocampal neurons in vitro was established successfully. From the findings of morphological changes and apoptosis rate of neurons, the oxygen glucose deprivation for 6 hours may be the suitable length of time for inducing neuron injury in this model.