中等强度磁场对神经元离子通道特性的影响及其机制探讨
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摘要
随着各种通信设备、家用电器的日益普及和各种理疗设备的应用,人类受到电磁场或利或弊的影响越来越多,而对电磁场的生物效应的研究报道也日益增多,但其内在作用机理尚不清楚,仍需进行深入的实验和理论探索。因而,有必要研究不同参数的电磁场对人体的影响和作用,特别是在细胞层次上的探讨,将有助于排除以往流行病学方式研究中的个体差异的影响,更有利于在深层次上揭示电磁场对生物的作用机制。本研究利用全细胞膜片钳技术,从离子通道特性及细胞电活动的角度研究不同场强、频率和暴露时间的磁场对动物神经元通道特性的影响,包括磁场对小鼠神经元电压门控钠、钾通道电流的时间、电压、磁场强度和频率依赖性以及磁场对通道稳态激活和失活动力学特征的影响,这可在细胞和分子水平上探索磁场对动物神经元的影响和作用规律。本文的主要研究内容包括:
1.在细胞水平上,利用膜片钳技术研究恒定磁场的作用对细胞形成全细胞模式之后的存活时间(也即细胞的存活能力)的影响。在30mT和100mT恒定磁场的暴露过程中,记录神经元的钠通道电流情况。实验显示30mT恒定磁场可显著延长神经元的平均存活时间,平均存活时间可达对照组的一倍以上,而100mT恒定磁场暴露组细胞的存活时间与对照组没有显著性变化。
2.针对以往磁场对生物组织的影响多数停留在动物整体和器官水平上,利用膜片钳技术研究了一系列有代表性的3mT、30mT、100mT恒定磁场和3mT工频磁场对小鼠前额叶皮层神经元钠离子通道特性的影响。实验得出,不同强度和频率的磁场对神经元钠通道的激活和失活特性都产生了不同程度的影响,具有电压、时间以及磁场强度和频率依赖性。
3.研究了一系列磁场(3mT、30mT、100mT恒定磁场和3mT工频磁场)对小鼠前额叶皮层神经元瞬时外向钾通道和延迟整流钾通道特性的影响。结果表明不同参数的磁场对神经元两种钾通道特性产生了不同程度的影响。
4.针对目前动物标本的选择主要集中在大鼠上,而小鼠本身又有其无法取代的优点,通过多次的实验获得一种简单快速,能够制备大量小鼠神经元的急性分离方法。该方法获得的神经元具有良好的形态学特征和典型的电生理学特性,可用于膜片钳实验,这拓宽了细胞标本的选择。
With the increasingly popularity of various of communicating devices, household appliances and the application of many physiotherapy equipments, people are suffering more and more from the effects of electromagnetic fields. There are many research about the biological effects of electromagnetic fields, however, its mechanism is unclear, so it needs to be further explored. Therefore, it is necessary to study the influences and functions of the electromagnetic fields with different parameters on human being, especially on the cellular level, which will help to remove the effect of individual difference in the previous epidemiological study, and be more beneficial to reveal the biological mechanism of electromagnetic fields at a deeper level. From the aspects of the characters of ion channels and the electric activity of the cells, the influence of the magnetic field of different intensity, frequency and effect time to the channel characters of the animal neurons were researched using whole-cell patch-clamp technique. In this thesis, the dependences of the time, voltage, magnetic field intensity and frequency of the magnetic fields to the sodium and potassium channel currents of the neurons of the mouse and the influences of the magnetic fields to the activation and inactivation characteristics of the channels were researched. This will be able to explore the effects and rules of magnetic fields on animal neurons at cellular and molecular level.
The main research contents of the dissertation are as follows:
1. At the cellular level, using patch-clamp technique, we investigated the effects of static magnetic fields on the survival time (i.e. the viability of the cell) after the formation of the whole-cell mode. In the exposure process of 30mT and 100mT static magnetic field, the sodium channel currents were recorded. The experiments revealed that 30mT static magnetic field could increase the average survival time of the neurons one time longer than the cell which was not exposed under magnetic field. However, the average survival time of neurons exposed under the 100mT static magnetic field had no significant differences with the control group.
2. Most of the previous researches about the effects of biological tissues inflicted by magnetic fields are at the levels of entire body or organs. The effects of 3mT, 30mT, 100mT static magnetic fields and 3mT power frequency magnetic fields on the properties of sodium channel in cortex neurons of a mouse were studied using the patch-clamp technique at the level of cells in this thesis. The results revealed that the magnetic fields of different intensities and frequencies had different influences on the activation and inactivation properties of neuronal sodium channel with the dependences of voltage, time, intensity and frequency of magnetic field.
3. The effects of series of magnetic fields (3mT, 30mT, 100mT static magnetic field and 3mT power frequency magnetic field) were studied on the properties of transient outward potassium channel and delayed rectifier potassium channel of cortex neurons. The results show that the magnetic fields of different parameters produced different effects on two potassium channel properties of neurons.
4. At present, most researches choose the rat as the animal specimens, but the mouse have their own advantages can not be replaced by the rat. A simple and fast acute isolation method preparing a large number of neurons was obtained by a lot of experiments in this paper. The neurons obtained by this method have good morphological and typical electrophysiological characteristics, which can be used for patch-clamp experiments. Thus the method can broaden the choice of cell samples.
1.在细胞水平上,利用膜片钳技术研究恒定磁场的作用对细胞形成全细胞模式之后的存活时间(也即细胞的存活能力)的影响。在30mT和100mT恒定磁场的暴露过程中,记录神经元的钠通道电流情况。实验显示30mT恒定磁场可显著延长神经元的平均存活时间,平均存活时间可达对照组的一倍以上,而100mT恒定磁场暴露组细胞的存活时间与对照组没有显著性变化。
2.针对以往磁场对生物组织的影响多数停留在动物整体和器官水平上,利用膜片钳技术研究了一系列有代表性的3mT、30mT、100mT恒定磁场和3mT工频磁场对小鼠前额叶皮层神经元钠离子通道特性的影响。实验得出,不同强度和频率的磁场对神经元钠通道的激活和失活特性都产生了不同程度的影响,具有电压、时间以及磁场强度和频率依赖性。
3.研究了一系列磁场(3mT、30mT、100mT恒定磁场和3mT工频磁场)对小鼠前额叶皮层神经元瞬时外向钾通道和延迟整流钾通道特性的影响。结果表明不同参数的磁场对神经元两种钾通道特性产生了不同程度的影响。
4.针对目前动物标本的选择主要集中在大鼠上,而小鼠本身又有其无法取代的优点,通过多次的实验获得一种简单快速,能够制备大量小鼠神经元的急性分离方法。该方法获得的神经元具有良好的形态学特征和典型的电生理学特性,可用于膜片钳实验,这拓宽了细胞标本的选择。
With the increasingly popularity of various of communicating devices, household appliances and the application of many physiotherapy equipments, people are suffering more and more from the effects of electromagnetic fields. There are many research about the biological effects of electromagnetic fields, however, its mechanism is unclear, so it needs to be further explored. Therefore, it is necessary to study the influences and functions of the electromagnetic fields with different parameters on human being, especially on the cellular level, which will help to remove the effect of individual difference in the previous epidemiological study, and be more beneficial to reveal the biological mechanism of electromagnetic fields at a deeper level. From the aspects of the characters of ion channels and the electric activity of the cells, the influence of the magnetic field of different intensity, frequency and effect time to the channel characters of the animal neurons were researched using whole-cell patch-clamp technique. In this thesis, the dependences of the time, voltage, magnetic field intensity and frequency of the magnetic fields to the sodium and potassium channel currents of the neurons of the mouse and the influences of the magnetic fields to the activation and inactivation characteristics of the channels were researched. This will be able to explore the effects and rules of magnetic fields on animal neurons at cellular and molecular level.
The main research contents of the dissertation are as follows:
1. At the cellular level, using patch-clamp technique, we investigated the effects of static magnetic fields on the survival time (i.e. the viability of the cell) after the formation of the whole-cell mode. In the exposure process of 30mT and 100mT static magnetic field, the sodium channel currents were recorded. The experiments revealed that 30mT static magnetic field could increase the average survival time of the neurons one time longer than the cell which was not exposed under magnetic field. However, the average survival time of neurons exposed under the 100mT static magnetic field had no significant differences with the control group.
2. Most of the previous researches about the effects of biological tissues inflicted by magnetic fields are at the levels of entire body or organs. The effects of 3mT, 30mT, 100mT static magnetic fields and 3mT power frequency magnetic fields on the properties of sodium channel in cortex neurons of a mouse were studied using the patch-clamp technique at the level of cells in this thesis. The results revealed that the magnetic fields of different intensities and frequencies had different influences on the activation and inactivation properties of neuronal sodium channel with the dependences of voltage, time, intensity and frequency of magnetic field.
3. The effects of series of magnetic fields (3mT, 30mT, 100mT static magnetic field and 3mT power frequency magnetic field) were studied on the properties of transient outward potassium channel and delayed rectifier potassium channel of cortex neurons. The results show that the magnetic fields of different parameters produced different effects on two potassium channel properties of neurons.
4. At present, most researches choose the rat as the animal specimens, but the mouse have their own advantages can not be replaced by the rat. A simple and fast acute isolation method preparing a large number of neurons was obtained by a lot of experiments in this paper. The neurons obtained by this method have good morphological and typical electrophysiological characteristics, which can be used for patch-clamp experiments. Thus the method can broaden the choice of cell samples.
引文
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