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目的探讨N-甲基-D-天门冬氨酸(NMDA)诱导的小脑颗粒神经元(CGNs)钙超载与NMDA受体(NMDAR)及细胞内钙库受体三磷酸肌醇受体(IP3R)和兰尼定受体(RyR)之间的关系。方法取出生后8 d SD大鼠小脑进行颗粒神经元体外培养。用NMDA(100μmol/L)急性损伤神经元,在激光扫描共聚焦显微镜(LSCM)扫描开始前30 min,或扫描开始后4,9,14 min分别加入NMDAR、IP3R、RyR拮抗剂MK-801、2-APB和DAN,检测神经元内Ca2+浓度的动态变化。结果 MK-801预孵育神经元经NMDA急性刺激后,神经元内Ca2+的荧光强度不再升高,NMDA刺激后加入MK-801,上升的Ca2+水平立即下降,最终下降至基线水平;NMDA急性刺激2-APB预孵育神经元,神经元内Ca2+的荧光强度升高,但升高幅度明显低于未经NMDA刺激组,NMDA刺激后加入2-APB,细胞内升高的Ca2+水平急剧下降,最终下降至接近基线水平;DAN预孵育的神经元经NMDA急性刺激后,胞内Ca2+的荧光强度急剧升高,达到NMDA刺激组水平,NMDA刺激后加入DAN,神经元内Ca2+水平无明显下降。结论 NMDA诱导的神经元Ca2+超载,主要由细胞膜钙通道NMDAR和细胞内钙释放通道IP3R介导,而细胞内钙释放通道RyR不起主导作用。
Objective To investigate the effects of N-methyl-D-aspartate (NMDA) -induced calcium overload on cerebellar granule neurons (CGNs) and NMDA receptor (NMDAR) and intracellular calcium receptor IP3R And Ryanidine receptor (RyR) between the relationship. Methods The cerebellar granule neurons were cultured in vitro on the 8th day after birth in SD rats. The neurons were injured acutely with NMDA (100μmol / L). NMDAR, IP3R and RyR antagonist MK-801 were respectively added 30min before laser scanning confocal microscopy (LSCM) scan and 4,9,14min after scan start. 2-APB and DAN to detect the dynamic changes of Ca2 + concentration in neurons. Results After MK-801 pre-incubated neurons were stimulated by NMDA, the fluorescence intensity of Ca2 + in neurons was no longer increased. After MK-801 was added into NMDA-stimulated cells, the ascending Ca2 + level decreased immediately and then dropped to the baseline level. 2-APB pretreatment neurons, the intracellular Ca2 + fluorescence intensity increased, but the magnitude of increase was significantly lower than without NMDA stimulation group, NMDA stimulation added 2-APB, intracellular elevated Ca2 + levels dropped sharply, and ultimately Decreased to close to the baseline level. The NMDA-stimulated fluorescence intensity of DAN preincubated neurons increased sharply after NMDA stimulation, reaching the level of NMDA stimulated group. After NMDA stimulation, the intracellular Ca2 + level was not significantly decreased. Conclusion NMDA-induced neuronal Ca2 + overload is mainly mediated by the membrane calcium channel NMDAR and the intracellular calcium release channel IP3R, whereas the intracellular calcium release channel RyR does not play a dominant role.