海藻糖对冻存海马神经元电生理特性的影响

Effect of trehalose on electrophysiological characteristics of cryopreserved hippocampal neurons

目的:观察原代海马神经元在不同条件下进行冷冻保存,经复苏后海马神经元电生理特性的改变。方法:将原代体外培养的海马神经元和培养当天经无血清冻存液、DF12+10%DMSO冻存液、DF12+10%DMSO+海藻糖冻存液冻存后复苏的海马神经元,在体外培养14~16 d。利用膜片钳技术记录各组动作电位、微抑制性突触后电流(mIPSC)、微小兴奋性突触后电流(mEPSC)。结果:使用含海藻糖的冻存液冻存过的海马神经元与未冻存原代培养海马神经元电生理特点接近;而经无血清冻存液冻存的神经元复苏后存在明显的Burst样放电(P<0.05),mIPSC幅度减小(P<0.001);经DF12+10%DMSO冻存液冻存的神经元复苏后存在明显的Burst样放电(P<0.05),mIPSC幅度减小(P<0.001)、频率减小(P<0.01)。结论:海藻糖对冻存海马神经元复苏后功能具有保护作用。

Objective:To observe the changes in the electrophysiological characteristics of primary hippocampal neurons after cryopreservation under different conditions.Methods:Primary hippocampal neurons cultured in vitro and hippocampal neurons recovered after being frozen in serum-free cryo-medium,DF12+10%DMSO cryo-medium,DF12+10%DMSO+trehalose cryo-medium on the day of culture.After culturing for 14 to 16 days,the action potential,miniature inhibitory postsynaptic current(mIPSC),and miniature excitatory postsynaptic current(mEPSC)of each group were recorded by patch-clamp.Results:Electrophysiological characteristics of hippocampal neurons cryopreserved in cryopreservation fluid containing trehalose were similar to those of unfrozen primary culture hippocampal neurons.However,neurons cryopreserved in serum-free cryo-medium had obvious Burst-like discharge after resuscitation(P<0.05),the amplitude of mIPSC was weakened(P<0.001);similarly,neurons cryopreserved in DF12+10%DMSO cryo-medium also had obvious Burst-like discharge(P<0.05),the amplitude of mIPSC was weakened(P<0.001),and the frequency of mIPSC decreased(P<0.01).Conclusions:Trehalose has a protective effect on the frozen hippocampal neurons after resuscitation.