缝隙连接蛋白Cx32和Cx43在大鼠癫癇样放电后神经元的表达及生胃酮的干预作用

Expression of gap junction connexin 32 and connexin 43 and inhibition of carbenoxolone in cultured hippocampal neurons of rats after epileptiform discharges

目的研究缝隙连接在癫发病机制中的作用。方法以体外培养大鼠海马神经元为研究对象,采用免疫细胞化学方法和实时定量逆转录-聚合酶链反应(RT-PCR)方法观察缝隙连接蛋白(connexin,Cx)32和Cx43的表达,并加用生胃酮干预。结果神经元经无镁液处理1h后Cx32mRNA迅速升高,至5h升高了近10倍;蛋白表达在2h后开始增多(21.80±1.74),8h后(47.30±5.75)较对照组(9.30±1.25)增高了5倍。Cx43水平低于Cx32,无镁液作用5h后mRNA表达显著增多,8h后蛋白表达始明显增强。生胃酮显著抑制了其表达。结论Cx32和Cx43在癫样放电后表达显著增多,生胃酮能够抑制其表达和神经元放电,提示缝隙连接在癫的发生发展过程中具有重要的作用。

Objective To study the role of gap junctions in epileptiform activity. Methods The epileptiform activity was induced by zero-Mg^2＋ medium in cultured hippocampal neurons of newborn rats. Immunocytochemistry and real time RT-PCR were introduced to evaluate the expression of gap junction Cx32 and Cx43. Results The level of Cx32 mRNA increased quickly one hour after the neurons were treated with zero-Mg^2＋ medium and was raised by 10 times 5 hours later, while Cx32 protein began to develop at the 2nd hour （21.80 ± 1.74） and was raised by 5 times at the 8th hour （47.30 ± 5.75 ）. The expression of Cx43 mRNA went up obviously 5 hours later, and Cx43 protein developed visibly 8 hours later. Carbonoxolone depressed the expressions of Cx32 and Cx43. Conclusions The expression of Cx32 and Cx43 increases dramatically after epileptic discharges and carbenoxolone inhibits both the discharges and the expression of gap junctions, which indicates that gap junction could contribute to epileptogenesis.