Munc13-1及RIM1在颞叶癫痫患者及戊四氮大鼠模型中的表达

目的 通过研究神经突触前膜胞内蛋白13-1(mammalian uncoordinated 13-1,Munc13-1)及神经元突触膜胞外分泌调节蛋白1(rab-interacting molecules 1,RIM1)在颞叶癫痫患者皮质和戊四氮点燃癫痫大鼠模型中的表达变化及两者的相关性,初步探讨Munc13-1及RIM1在癫痫发生中的作用及机制.方法 以颞叶癫痫患者病灶切除的皮质为癫痫组(n=18),以脑外伤患者切除的正常皮质为对照组(n=15);取40只SPF级雄性SD大鼠抽签均分成2组,其中一组连续腹腔注射戊四氮(3.5 mg/kg)21 d,出现至少连续3dⅣ级及以上癫痫发作的大鼠为癫痫组(n=15),另一组注射等量生理盐水为对照组(n=20);分别采用免疫组化及免疫荧光技术对Munc13-1进行定位和表达分析,Western blot检测Munc13-1和RIM1的表达变化,免疫共沉淀验证Munc13-1和RIM1的相互作用.结果 ①免疫荧光定位结果显示Munc13-1主要表达于神经元;②免疫组化及Western blot结果显示:在癫痫组中Munc13-1和RIM1的表达均升高,差异有统计学意义(P＜0.01);③免疫共沉淀示Munc13-1与RIM1之间存在相互作用的关系.结论 Munc13-1及RIM1在癫痫组中的表达均明显升高,且两者间存在相互作用的关系,提示Munc13-1可能通过结合RIM1参与癫痫的发生.

Expression of mammalian uncoordinated 13-1 and rab-interacting molecules 1 in temporal lobe epilepsy patients and pentetrazole-induced rat model

Objective To explore the roles and underlying mechanisms of mammalian uncoordinated 13-1 (Munc13-1) and rab-interacting molecules 1 (RIM1) in epileptogenesis through detecting the expression and interaction of the 2 molecules in the brain tissues of temporal lobe epilepsy patients and pentetrazoleinduced rats.Methods The excision of an epileptic focus from temporal lobe epilepsy patients were taken as the epilepsy group (n =18),and the normal cortex excisions of brain trauma patients were taken as the control group (n =15).Forty healthy male rats of SPF grade were randomly divided into pentetrazole group (3.5 mg/kg for 21 d),and control group (normal saline,n =20).The rats displaying epileptic seizure above grade Ⅳ for 3 consecutive days were selected as the epilepsy model group (n =15).The expression of Munc13-1 in the human cortex,rat cortex and rat hippocampus was detected by immunofluorescence staining and immunohistochemical assay.The expression level of Munc13-1 and RIM1 was detected by Western blotting.The interaction between the 2 molecules was verified by co-immunoprecipitation (Co-IP).Results ①Immunofluorescence staining showed that Munc13-1 was mainly located in the neuron.②Immunohistochemical assay and Western blotting showed that the expression of both Munc13-1 and RIM1 were significantly up-regulated in the epilepsy group (P ＜ 0.05).③Co-IP results indicated that there was evident interaction between the 2 molecules.Conclusion Munc13-1 and RIM1 are both up-regulated and interacted with each other in the epilepsy cortex,suggesting that Munc13-1 may be involved in epileptogenesis by binding to RIM1.