无镁诱导神经元放电后细胞共培养微环境中脑源性神经营养因子的变化

Level of brain-derived neurotrophic factor in the microenvironment of the neuron-astrocyte co-culture system by Mg^(2+)-free-induced seizure-like discharge

目的无镁诱导体外培养神经元(neurons,N)放电,研究放电后N和星形胶质细胞(astrocytes,AST)共培养体系中脑源性神经营养因子(BDNF)的改变并分析其主要来源。方法以纯化的胎鼠海马N和新生鼠AST为研究对象,按照是否经过短暂的无镁标准细胞外液处理诱导反复的自发性惊厥样放电,分为N对照组、N无镁组、N+AST对照组和N+AST无镁组。ELISA法测定惊厥后不同时间点细胞培养上清液中的BDNF含量。结果 N+AST无镁组恢复正常培养后48 h,AST的体积增大,突起及细胞数目增多。N无镁组恢复正常培养后72 h,仍旧存在自发性"惊厥样放电"。N+AST对照组在24 h、48 h细胞上清液中的BDNF浓度较N对照组同时间点细胞上清液中BDNF升高(P﹤0.05)。N+AST无镁组在12 h、24 h、48 h分泌的BDNF均较N+AST对照组相同时间点的BDNF升高,尤其12 h、24 h升高明显(P﹤0.01);而N无镁组在各个时间点BDNF含量也有升高,但与N对照组比较,差异无统计学意义。N+AST无镁组在24 h分泌的BDNF较N无镁组同时间点分泌的BDNF升高(P﹤0.05)。结论 N+AST在正常培养情况下分泌的BDNF由N和AST共同参与,N可能起主要分泌作用。短暂无镁处理的N+AST,经诱导放电后BDNF的分泌显著增多,并且可能主要来自惊厥微环境活化的AST。

Objective To study the level of brain-derived neurotrophic factor（BDNF） in the microenvironment of the neuron-astrocyte co-culture system by Mg^2＋ -free-induced seizure-like discharge and analyze the source of BDNF. Methods Hippoeampal neurons （N） of fetal rats and astrocytes （AST） of neonatal rats were purified and divided into four groups, included control N （ Con N） group, Mg^2＋ -free treated N （ Mg^2＋ -free N） group, control N ＋ AST co-culture （ Con N ＋ AST） group and Mg^2＋ -free treated N ＋ AST co-culture（ Mg^2＋ -free N ＋ AST） group. The Mg^2＋ -free treated groups were exposed to Mg^2＋-free media for 3 hrs to induce a repeated spontaneous seizure-like discharge. The level of BDNF in each group at different time points was measured using ELISA. Results The cellular morphous of AST changed in the Mg^2＋ -free N ＋ AST group at 48 hrs. Neuronal epileptiform activity was observed in the Mg^2＋ -free media at 3 hrs, and continued to exist until the mieroenviornment returned to normal for 72 hrs. The BDNF level increased at 24 hrs and 48 hrs in the Con N ＋ AST group compared with the control N group （ P 〈 0.05 ）. Compared with Con N ＋ AST group, BDNF level increased at 12, 24 and 48 hrs in the Mg2^-free N ＋ AST group, especially at 12 and 24 hrs （P 〈 0.01 ）. There were no significant differences in the level of BDNF between the Con and Mg^2＋ -free N groups. Compared with Mg^2＋ -free N group, BDNF level increased at 24 hrs in the Mg^2＋ -free N ＋ AST group （P 〈 0. 05 ）. Conclusions The results of the experiment suggest that BDNF in the Con N ＋ AST group might be excreted from both N and AST, but chiefly from N. Activated AST may be the main source for increasing BDNF in the Mg^2＋ -free N ＋ AST group.