体外氧糖剥夺再复氧大鼠皮层神经元细胞活性及EphA4蛋白的表达

Detection of EphA4 and cell viability in rat cortical neurons during oxygen and glucose depriration and reoxygen in vitro

目的:观察EphA4在氧糖剥夺再复氧体外培养大鼠皮层神经元的表达变化及可能的作用。方法:体外培养新生0～1d的Wistar大鼠皮层神经元,分为对照组和氧糖剥夺(OGD)再复氧模型组。模型组通过更换培养基为无糖Earle液并通入体积分数95%N2/5%CO2混合气体建立OGD模型,4h后复氧。选取复氧0、4、12和24h4个时间点,四唑盐(MTT)试验观察神经元活性,免疫细胞化学方法检测EphA4蛋白的表达情况。结果:模型组4个时间点细胞活性均低于对照组(t=19.556,20.909,13.673,16.737,P均<0.01),且随着复氧时间延长,活性逐渐降低(F=573.510,P<0.01)。再复氧4、12及24h,模型组EphA4表达较对照组明显升高(t=1.524,6.821,7.950,11.593,P<0.01),并随复氧时间延长有逐渐增高趋势(F=40.205,P<0.01)。结论:在缺血再灌注过程中,EphA4可能参与了抑制神经再生。

Aim ： To explore the expression and possible role of EphA4 in the cultured rat cortical neurons from rats with oxygen and glucose deprivation （OGD） and reoxygen model. Methods ： Cortical neurons of Wistar rats born within 1 day were cultured in vitro. The neurons were divided into 2 groups,control group and model group. The model was established by replacing the medium with Earleg fluid without glucose and fluxing a gas mixture consisting 95% N2/5% O5 , 4 hours after OGD,reoxygen was administered. Cell viability was tested by MTT assay and EphA4 expression assessed by immunocytochemistry method at 0,4,12 and 24 h after reoxygen in both groups. Results ： There was a great decrease of cell viability in model group compared with that of control group（ t = 19. 556,20. 909,13. 673,16. 737 ,P 〈 0.01 ）. Cell viability in model group decreased with reoxyen time （F = 573.510, P 〈 0.01 ）. There was a significant difference in the expression of EphA4 between the two groups at 4,12 and 24 h （ t = 1. 524,6.821,7. 950,11. 593, P 〈 0.01 ）. In model group, the expression of EphA4 increased with reoxyen time （ F = 40. 205, P 〈 0.01 ）. Conclusion ： In ischmic and reperfusion course, EphA4 may exihit inhibitory role in neural regeneration.