海马过表达Ephrin-B3对颞叶癫痫大鼠突触重塑的影响

Effect of overexpression of tyrosine protein kinase B3 in rat hippocampus on synaptic remodeling of temporal lobe epilepsy

目的探讨海马内过表达酪氨酸蛋白激酶B3(Ephrin-B3)对大鼠癫痫突触重塑的可能作用机制。方法将40只雄性SD大鼠随机分为空白对照组、癫痫组、空载体组、慢病毒Efnb3过表达组,每组10只。除空白组外,其余3组均进行癫痫造模处理,造模前1周空载体组两侧海马各注射LV5-NC(5μL)载体,慢病毒过表达组海马注射包装后的Efnb3慢病毒(5μL)进行预处理。观察各组大鼠行为学变化,癫痫造模达24 h后各组取海马组织,采用qPCR检测Ephrin-B3、突触后密度蛋白95(PSD95)、离子型谷氨酸受体亚基(NR2B)的mRNA相对表达变化,蛋白质印迹法检测Ephrin-B3、PSD95、NR2B的蛋白相对表达量。结果Ephrin-B3过表达组癫痫发作潜伏期(30.2±4.38)min,较癫痫组(22.4±3.91)min和空载体组(21.0±5.29)min有所延长(P<0.05),癫痫组和空载体组造模后Ephrin-B3、PSD95、NR2B的mRNA表达量降低,转染Ephrin-B3成功组mRNA相对表达量相较于癫痫模型组明显增加(Ephrin-B3:F=25.11,P=0.0027;PSD95:F=14.80,P=0.0203;NR2B:F=19.51,P=0.0010),相较于空载体注射组亦明显增加(Ephrin-B3:P=0.0029;PSD95:P=0.0160;NR2B:P=0.0034);转染Ephrin-B3成功组相较于癫痫模型组蛋白相对表达量增加(Ephrin-B3:F=17.72,P=0.0032;PSD95:F=7.889,P=0.0145;NR2B:F=9.755,P=0.0199),较空载体注射组表达亦明显增加(Ephrin-B3:P=0.0034;PSD95:P=0.0253;NR2B:P=0.0144)。结论过表达Ephrin-B3可减轻癫痫发作损伤,其机制可能与调控突触后蛋白PSD95、NR2B的表达量控制突触重塑有关。

Objective To investigate the possible mechanism of overexpression of tyrosine protein kinase B3(Ephrin-B3)in the hippocampus on synaptic remodeling in rats with epilepsy.Methods Forty male SD rats were randomly divided into blank control group,epilepsy group,empty carrier group and lentivirus Efnb3 overexpression group,with 10 rats in each group.Epilepsy modeling was performed in all the other three groups except the blank group.One week before modeling,LV5-NC(5μL)vector was injected into the hippocampus on both sides of the empty vector group,and Efnb3 lentivirus(5μL)was injected into the hippocampus of the lentivirus overexpression group.Ephrin-B3,postsynaptic densitin-95(PSD95)and ion glutamate receptor subunit(NR2B)were detected by qPCR from hippocampal tissues taken from each group 24 hours after the seizure model,while the expressions of PSD95 and NR2B were detected by western blotting.Results The seizure latency of Ephrin-B3 overexpression group(30.2±4.38)minutes was significantly longer than that of Ephrin-B3 overexpression group(22.4±3.91)minutes and empty vector group(21.0±5.29)minutes(P<0.05).The mRNA tables of Ephrin-B3,PSD95 and NR2B in the epileptic and empty vector groups after the model were constructed.The mRNA relative expressions of Ephrin-B3 transfected group were significantly increased compared with those of the epilepsy model group(Ephrin-B3:F=25.11,P=0.0027;PSD95:F=14.80,P=0.0203;NR2B:F=19.51,P=0.0010)and empty vector group(Ephrin-B3:P=0.0029;PSD95:P=0.0160;NR2B:P=0.0034).The relative expressions of histone in Ephrin-B3 transfected group increased compared with those in the epileptic model(Ephrin-B3:F=17.72,P=0.0032;PSD95:F=7.889,P=0.0145;NR2B:F=9.755,P=0.0199)and empty vector group(Ephrin-B3:P=0.0034;PSD95:P=0.0253;NR2B:P=0.0144).Conclusion Overexpression of Ephrin-B3 can alleviate the injury of epileptic seizure,which may be related to regulating the expression of postsynaptic proteins PSD95 and NR2B to control synaptic remodeling.