电刺激对背根节神经元/Schwann细胞联合培养体系髓鞘蛋白P0表达的影响

Effect of electrical stimulation on the expression of myelin protein zero P0 from DRGN/Schwann cell co-culture in vitro

目的:建立体外背根节神经元与Schwann细胞联合培养模型,观察短时低频电刺激对Schwann细胞髓鞘蛋白表达的影响。方法:培养和纯化背根节神经元与Schwann细胞,制成背根节神经元/Schwann细胞联合培养体系。于L-ascorbic acid诱导的同时,施予低频电刺激(20Hz,100μs,3V),持续作用1h,分别于L-ascorbicacid诱导后第0,2,4,8和10d取各组培养基上清以ELISA测定其中脑衍生物神经生长因子(BDNF)的水平。另外,于诱导后第7d和14d检测培养体系中髓鞘蛋白P0的表达。结果:电刺激组各时间点BDNF的浓度较对照组显著升高(P<0.01)。经电刺激作用后,联合培养体系中P0表达上调(P<0.05)。然而,电刺激结束后在培养液中加入TrkB-Fc,P0的表达水平则显著降低(P<0.05)。结论:在神经元存在的条件下,短时低频电刺激可促进离体Schwann细胞合成P0,初步认为该作用通过刺激神经元分泌BDNF增多所致。

Objective：To establish the co-culture model of dorsal root ganglion neurons（DRGNs）and Schwann cells（SCs）,and to investigate the effect of electrical stimulation（ES）on myelin protein expression by SCs.Methods：Purified DRGNs and SCs were co-cultured and subjected to continuous ES（20 Hz,100 μs,3V）for one hour at the time point of L-ascorbic acid addition.The conditioned supernatants of cell cultures were collected for measuring the concentration of brain-derived neurotrophic factor（BDNF）by ELISA at 0,2,4,8 and 10 days after L-ascorbic acid induction.At 7 and 14 days after induction,the expression of myelin protein P0 in each group was examined by Western Blot.Results：BDNF secretion（P〈0.01）and P0 expression（P〈0.05）in the ES-treated groups was dramatically enhanced as compared with the control groups.However,P0 expression from the ES-treated groups reduced significantly by using the BDNF scavenger TrkB-Fc after ES treatment（P〈0.05）.Conclusion：In the presence of neurons,short-duration low-frequency ES promoted P0 expression by SCs,and its effect was at least initially mediated via the enhancement of BDNF release from neurons.