摘要
电气化是人类文明进程的里程碑。然而越来越多的研究提示，极低频电磁场可引起有害的生物效应。特别是有研究报道，由高压输电线等产生的工频电磁场可增加儿童白血病，神经系统肿瘤及淋巴瘤发病的危险率。为了揭示工频磁场生物效应的机制，尤其是与肿瘤发生发展的关系，人们开展了较为广泛的体内及体外的实验研究，涉及到各种生物酶类(ODC、琥珀酸脱氢酶、PKC等)、原癌基因(c-myc、c-fos等)、细胞间缝隙连接及蛋白合成等方面。然而极低频电磁场生物效应的机制仍不明确。
环境中极低频电磁场通常因能量较低而难以直接与生物大分子作用或导致DNA损伤，引发突变、畸变及断裂等效应。因此一般认为电磁场可能通过作用于细胞膜上业已存在的信号转导途径，将物理信号转化成生物信号，进而经过信号转导及生物级联放大，最终产生生物效应。对极低频电磁场信号转导过程的研究，有利于揭示其生物效应的机制。相关的研究表明，SAPK途径及P38MAPK途径是哺乳类动物细胞内普遍存在，并介导物理性应激的两条重要途径，它们通过蛋白质的级联磷酸化过程来进行信号的转导。极低频电磁场是环境中的一种物理应激因子，因此推测极低频电磁场的生物效应与该途径间存在一定的联系。所以本实验以蛋白质酪氨酸磷酸化为起点，研究极低频磁场对SAPK途径及P38MAPK途径的影响。细胞膜是电磁场信号的可能耦合位点，相关研究也证实了细胞膜受体在细胞转导物理性应激因子中的作用。因此本文又进一步探索了极低频磁场对细胞膜受体聚簇的作用。
近年来，Litovitz等提出了电磁场的时空相干理论：认为规则电磁场是相干场，可诱导生物体产生效应，而噪声磁场为非相干场，可以干预前者的生物效应。并以系列的实验验证了该理论。然而，由于验证该理论的实验研究所用的磁场强
浙江大学博士学位论文
度很低（微特斯拉级），其本身所诱导的生物学效应存在不确定性，科学界对其
结果尚存在争论，因此，噪声磁场的干预效应一直没有被广泛接受。我们认为，
从理论上分析其效应可能存在。故本文使用上述经反复验证的、强度 0．4 mT、
具有明确生物效应的工频磁场及相同强度的噪声磁场，以SAPK磷酸化及细胞膜
EGF与hF受体聚簇程度为研究指标，探索了噪声磁场及由噪声磁场与工频磁
场叠加产生的复合磁场的生物效应。
1．工频磁场对细胞蛋白质酪氨酸磷酸化的影响
蛋白质级联磷酸化是细胞内信号转导过程的主要方式，也是蛋白质活化的主
要方式。为了研究工频磁场对细胞蛋白质酪氨酸磷酸化的作用，将常规培养的中
国仓鼠肺成纤维细胞（CHL）分组并作不同处理：厂）．对照组，不作任何处理；
o）．0．4mT磁场辐照处理组，常规培养 3天后的细胞以 0．4mT频磁场辐照处理
不同时问；o）．0．smT磁场辐照处理组，常规培养 3天后的细胞以 0．smTI频磁
场辐照处理不同时间。用裂解液裂解细胞，提取蛋白质。定量后，以Western印
迹方法测定并比较细胞蛋白质酪氨酸磷酸化的程度。结果发现0、4mT和0．smT
两个强度的工频磁场均可影响细胞蛋白质酪氨酸磷酸化。其中，两个强度都能影
响分子量为38、97．4kDa蛋白质的酪氨酸磷酸化。此外，0．4mT强度还可使61．7、
105及112kDa的蛋白质酪氨酸磷酸化发生改变，而0．smT强度却使79、150kDa
的蛋白质酪氨酸磷酸化发生变化。并且上述蛋白质酪氨酸磷酸化的变化均存在着
时间相关性。
2．工频磁场对应激活化蛋白激酶信号途径的影响
应激活化蛋白激酶（SAPK）途径是细胞内转导外界物理性应激因于的重要
途径之一。本文将常规培养的 CHL细胞分别经下列处理：＊）．对照组，不作任
何处理；p）．0．4mT磁场辐照处理组，常规培养3天后的细胞以0．4mT工频磁场
辐照处理不同时间；O）．0．smT磁场辐照处理组，常规培养 3天后的细胞以 0．smT
工频磁场辐照处理不同时间。用裂解液裂解细胞，提取蛋白质，定量。利用Western
印迹方法，分别以 Anti－JNKI（FL）mana Cruz＼ MEK4u）抗体（Santa
Cruz）及 Phospho－SEKI／MKK4（hr233）抗体（BioLabs），识别 JNK，SEKI及
磷酸化的SEKI，并通过JNK的滞后条带识别磷酸化的JNK条带。比较辐照前
后细胞中 SAPK的磷酸化程度及 SEK MKK4活化程度。随后以固相激酶分析法
－3－
浙江大学博士学位论文
＊olidphase kinase assny人 对分别经两个强度辐照15 min后的细胞SAPK活性
进行测定。结果提示 CHL细胞经 0人及 0．8 mT频磁场辐照处理后，可呈时相
性增强 SAPK磷酸化，并且均在 15 min时达到最大值，SAPK磷酸化分别提高
20％和 17％；同时 SAPK激酶活性也相应增强，分别是对照的 2．9士 0．4和二＊士
0．9倍。然而，0．8 mT诱导 SAPK磷酸化的时程长于 0．4 mT，而脱磷酸化的时程
短于 0．4 mT。SAPK上游激酶 SEKI／MKK4的磷酸化却不受工频磁场的影响。
为了探索工频磁场诱导SAPK激酶活化过程与PKC激酶之间的关系，我们
用PKC的特异性抑策剂（Protein Kinase C Inhibitof3 EGF－R Fragment 651－65
Electrification is the landmark in the civilization of mankind. However, evidence is accumulating that exposure to extremely low frequency electromagnetic field may produce a lot of biological effects. Especially, some reports showed that ELF-EMF such as those from electric power transmission and distribution lines have been associated with increased risk of childhood leukemia, cancer of the nervous system, and lymphomas. In order to find an explanation and to determine the possible mechanism of these biological effects, many in vitro and in vivo studies of EMF effects have been published. These studies were involved in many enzymes (such as ODC, succinate dehydrogenase, PKC), pro-oncogene (c-myc, c-fos, etc.), gap junction and protein synthesis, and so on. But the mechanism of the biological effects of ELF-EMF is still unclear.
Low-frequency electromagnetic fields in our environment often have little energy to directly traverse the membrane, and act with biological molecules, or damage the DNA. It is possible that they may modify the existing signal transduction procession in cell membrane, thus producing both transduction and biochemical amplification of the effects of the field itself. So the related mechanism studies of the cellular signal transduction have advantages for investigating biological effects of ELE-EMF. The related studies showed that the stress-activated protein kinase (SAPK) and P38 mitogen-activated protein kinase (P38 MAPK) signal transduction pathways are universally existing in the mammalian cells, which transduct the environmental
physical stresses into cell and produce biological effects through the cascade phosphorylation between proteins. ELF-EMF is also a physical factor. We suppose that these signal pathways may mediate 50Hz magnetic field (MF) signals in cells. The purposes of this study were to investigate the possible effects of exposure to 50Hz MF on protein tyrosine phosphorylation, SAPK signal pathway and P38 MAPK signal pathway. Cellular membrane is the possible action site for ELF-EMF, and the related studies confirmed that the receptors on cell membrane could be clustered by physical stress factor, and the receptors transduced the signal into cells via cellular signal pathway. Thus, we also investigated the effects of ELF-MF exposure on clustering of EGF, TNF receptors in cultured cells.
Recently, Litovitz et al raised the spatial coherence theory of electromagnetic field (EMF). The theory thinks that regular MF is spatial coherence field and noise MF is spatial incoherence field, the noise MF could inhibit the biological effects induced by MF. Following it, there are serial experiments to support this theory. However, the inhibition effects of noise MF on biological effects induced by MF were not accepted universally because the intensity of MF which used in these experiments was too small (at micro-Tesla order) to produce the certain biological effects. So the theory is controversy. We think the inhibition effects of noise MF may be existence at theory level. In order to confirm the effects of noise MF, in the present study, we use 0.4mT MF and noise MF, which intensity is hundred times than the previous studies and is proved repeatedly in our experiments to produce the clear biological effects, to examine the effects of noise MF on the clustering of EGF, TNF receptors and SAPK phosphorylation induced by 50Hz MF.
1. Effects of 50Hz Magnetic Field on Protein Tyrosine Phosphorylation in Cultured Cells
Protein cascade phosphorylation is one of the main modes of signal transduction pathway and protein activation in cells. In order to investigate the effects of 50Hz MF exposure on protein tyrosine phosphorylation, Chinese Hamster Lung (CHL) cells were cultured routinely 3 days in MEM medium. After treated as follows: (1). Control, without any treated; (2). 0.4mT MF exposure, exposed to 0.4mT MF for different
durations; (3). 0.8mT MF exposure, exposed to 0.8mT MF for different durations, the cells were harvested and protein was extracted. Western blotting
引文
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