依托咪酯对小鼠诱导分化神经元缝隙连接子表达量的影响

The influence of etomidate on connexons of induced differentiated neurons in mouse

目的观察全身麻醉药物依托咪酯对P19细胞诱导所获得的神经元缝隙连接子表达的影响,以揭示依托咪酯引起神经外科手术患者意识迅速丧失可能的分子学机制。方法 P19细胞经体外培养诱导分化为可特异性表达微管蛋白之神经元,免疫荧光细胞化学染色检测依托咪酯对神经元缝隙连接子表达的影响。结果依托咪酯和生胃酮处理P19细胞30 s和60 min后,免疫荧光细胞化学染色即可发现神经元缝隙连接子表达受到抑制,缝隙连接蛋白36(Cx36)阳性细胞数目显著减少,与对照组比较差异具有统计学意义(30 s:t=-50.423,P=0.000;60 min:t=-25.826,P=0.000);随着处理时间的延长,其抑制效应逐渐降低,与药物处理前比较差异无统计学意义(均P>0.05)。处理30 s时,依托咪酯和生胃酮对神经元缝隙连接子的抑制效应相似(P>0.05),而随着药物处理时间的延长,相同作用时间、相同作用对象依托咪酯对神经元缝隙连接子的抑制效应逐渐增强且优于生胃酮组,以处理60 min时二者差异最大(t=-2.782,P=0.008)。结论依托咪酯可能通过抑制神经元缝隙连接子的表达水平而降低神经元细胞间缝隙连接通讯,能够部分解释依托咪酯在麻醉诱导过程中引起患者意识迅速丧失的可能的分子学机制。

Objective To explore the effect of general anesthetic, etomidate, on the expression of connexons in neurons induced by P19 cells, and illustrate the molecular mechanism of patients consciousness loss after etomidate administration by means of immunucytochemistry. Methods The P19 cells were retro-induced to stem cells, which were then oriented-induced to neurons to receive neuron identification. The neurons were then allocated into group E （etomidate）, group CAR （carbenoxolone）, group LE （lipid emulsion） and group CON （phosphate buffer solution, control）. Agents administrated respectively were withdrawn in 30 s, 60 min and 120 min, and connexin36 （Cx36） were counted through inference of immunocytochemistry. Results Cx36 were more suppressed for group E and group CAR than group CON in 30 s and 60 rain （30 s： t = - 50.423, P = 0.000; 60 min： t = - 25.826, P = 0.000）. As for the inner group analysis, group E and group CAR bared declined suppressive effects with even no statistic variance in 120 rain compared to the respective untreated time points; as for group LE and group CON, no statistic variance existed between different times. Intergroup analysis showed that significant suppressive effects in 30 s were seen in group E and group CAR which were more than group LE and group CON （P 〈 0.05, for all） while the difference of suppressive effects were not seen between group E and group CAR （P 〉 0.05）. In 60 min,positive-stained eonnexons were more suppressed in group E than in group CAR （t = - 2.782, P = 0.008）. Etomidate gave more suppression than carbenoxolone and this stronger effect not shown until 60 min. Conclusion Etomidate depresses the gap junction intercellular connection （GJIC） by means of decreasing the number of neuron connexons, which might be the molecular mechanism for patients＂ consciousness loss after etomidate administration.