海马神经元ROS和Ca^(2+)对极低频电磁场实时响应的方法研究

Methodology Study on The Real-time Response of ROS and Ca^(2+) to Extremely Low Frequency Electromagnetic Fields in The Hippocampal Neurons

现有极低频电磁场(extremely low frequency-electromagnetic fields,ELF-EMFs)生物效应的研究方法是非实时组间对照,这种方法无法排除细胞对电磁场反应个体敏感性的差异,以及实验过程中条件变化的差异.本文提出一种对同一个细胞在同一条件下的前后对照实时观察ELF-EMFs反应的方法.采用稳定域鉴别ELF-EMFs暴露前海马神经元的稳定性,在电磁场加入时刻(t=60 s)分别用0、0.09、0.38、0.76、7.33、14.78 mT的ELF-EMFs作用于海马神经元,实时记录海马神经元活性氧(reactive oxygen species,ROS)和Ca^(2+)荧光响应曲线,建立ELF-EMFs与ROS和Ca^(2+)的自相关函数.结果表明:a.在ROS暴发时间和Ca^(2+)暴发时间,ROS和Ca^(2+)荧光响应具有阶跃性,这是判断实时响应的重要标志;b.ROS和Ca^(2+)对ELF-EMFs响应时间具有延时性,并且不一致;c.ROS和Ca^(2+)对ELF-EMFs实时响应具有剂量性;d.海马神经元ROS对ELF-EMFs响应具有复杂性;e.海马神经元Ca^(2+)对电磁场响应主要是渐进稳定.当ELF-EMFs与ROS和Ca^(2+)实时响应的相关函数大于0.3可以判定具有相关性.由此得出,一种ELF-EMFs暴露下细胞ROS和胞内Ca^(2+)的实时响应方法对电磁生物效应评价是可行的.

Existing research methods for biological effects of extremely low frequency electromagnetic fields(ELF-EMFs)are comparison between groups,with inability to exclude individual differences in cell sensitivity or changes in conditions during experiment.This work proposed a method for real-time effects observation of ELFEMFs in the same cell and same condition.The stability domain was used to identify the stability of hippocampal neurons before ELF-EMFs exposure.At the time of electromagnetic field intervention(t=60 s),ELF-EMFs were exposed to hippocampal neurons at 0,0.09,0.38,0.76,7.33 and 14.78 mT,respectively.Reactive oxygen species(ROS)and Ca^(2+)fluorescence response curves of hippocampal neurons were recorded in real-time,and the autocorrelation function between ELF-EMFs and fluorescence response was established.The results showed as follows:(1)The step properties of fluorescence response were clear at the burst times of ROS and Ca^(2+),which were important indicator to identify the real-time response;(2)The response time of ROS and Ca^(2+)to ELF-EMFs were delayed and inconsistent;(3)ROS and Ca^(2+)had dose-dependent response to ELF-EMFs;(4)The response of ROS to ELF-EMFs was complex;(5)The response of Ca^(2+)to ELF-EMFs was asymptotically stable.When correlation function exceeded 0.3,the real-time response of ELF-EMFs and ROS/Ca^(2+)was correlated.It was concluded that a real-time response method of intracellular ROS and Ca^(2+)to ELF-EMFs exposure was feasible for the evaluation of electromagnetic bioeffects.