电磁场对细胞蛋白质表达的影响

发布时间：2018-06-02 12:20

本文选题：电磁场 + 极低频电磁场　；参考：《浙江大学》2007年博士论文

【摘要】： 随着无线通讯技术和电力事业的飞速发展，电磁辐射已成为环境中增长最快、影响最为普遍的因素之一，对其健康危害的认识和预防事关我国科技、经济和社会的可持续发展。有流行病学调查显示极低频电磁场(ELF EMF)暴露可引起白血病和乳腺癌等发病率增高；移动电话的射频电磁场(RF EMF)暴露可影响中枢神经系统功能，导致脑瘤等恶性病变。这些流行病学调查结果推动了电磁场对生物体的生物学效应及其机理的研究。体内、体外的实验研究提示低强度电磁场对神经系统、生殖系统和免疫系统等可产生一定影响，但同时也有大量的阴性报道存在，导致无法对电磁场的健康危险度进行正确评估。造成这种现象的原因在于电磁场与生物体作用的原初物理过程、引发的生物学反应以及产生生物学效应的机制不清，电磁场生物学效应的研究存在一定的盲目性。因此，揭示低强度电磁场生物学效应及作用机制成为目前迫切需要解决的问题。生物系统受电磁场辐照所产生的各种生理生化改变可能涉及到基因的表达调控。有研究发现，电磁场可改变原癌基因、凋亡相关基因、周期调控基因等的mRNA水平，如极低频电磁场可诱导原癌基因c-myc、c-jun和c-fos的转录，改变鼠胚胎干细胞凋亡相关基因bcl-2和bax、细胞周期调控相关基因GADD45的转录：一定强度射频电磁场可下调神经元特异性Nurrl基因的表达，上调bax、GADD45 mRNA的水平；低频电磁场间断辐照可上调p53缺陷型细胞中c-jun、p21和egr-1 mRNA的水平，但野生型细胞不受影响：1710 MHz射频电磁场可显著上调p53缺陷型细胞中Hsp70 mRNA的转录，同时使c-jun、c-myc和p21 mRNA瞬时低幅度增加。基因在生物体的功能最终由其编码的蛋白质在细胞水平上体现，因此从蛋白质的角度入手才能真正揭示生命活动的规律。电磁场对细胞蛋白质表达的作用研究不多，主要集中在对鸟苷酸脱羧酶ODC、热休克蛋白HSP27／70以及一些信号转导途径中信号分子PKA、PKC、TPK、MAPK等表达水平或磷酸化等翻译后修饰的影响上。然而在这些研究中，实验者通常是根据推测的电磁场作用的可能效应、作用靶点和机制，选择相关的单个或几个蛋白质进行检测。这种研究思路是以假说为前提的，可能产生主观偏差；同时，由于所选择的指标是零散的，无法得到系统性、整体性的结果，不能全面揭示电磁场的生物学效应，勾画出其反应通路。一般认为，电磁场作为一种低能量的环境因素，可能通过复杂的信号传递过程作用于细胞，改变多个蛋白质的表达水平和／或翻译后修饰，进而产生一系列后续效应。因此，从蛋白质组的角度研究电磁场的生物学效应是必要的。以双向电泳作为分离技术和质谱作为鉴定技术的蛋白质组学方法能同时分离细胞内成百上千种蛋白质，并比较不同生理或病理状态下蛋白质表达的变化，为揭示外界因素对生物体的影响和疾病发生机制等提供了一种全新的研究方法。2001年，本实验室和芬兰Leszczynski研究小组率先将该技术引入到电磁场生物学效应及机制的研究中。为探讨电磁场对肿瘤发生的可能促进效应和比较环境中最常见的两类电磁场作用的异同，本博士论文第一部分选择人乳腺癌细胞株MCF-7，，采用双向电泳技术(2-DE)研究了50 Hz极低频磁场和1800 MHz射频电磁场对细胞蛋白质表达的影响，建立了该细胞的电磁场蛋白质差异表达图谱，进而利用质谱技术(MS)鉴定了部分电磁场反应蛋白质。根据第一部分的研究结果和一些文献的报道，我们认为有必要筛选确定电磁场作用敏感细胞，为此又设定了第二部分的研究内容。一般认为，电磁场对细胞的生物学效应受电磁场自身多因素的影响，如电磁场频率、强度、暴露时间和模式等。然而，相对于电磁场自身因素的影响而言，生物系统(细胞／组织等)的来源和辐照时的具体状态更能影响实验的最终结果。Leszczynski等发现SAR为2.4 W／kg的GSM 900射频场辐照EA．hv926细胞1小时可引起38个蛋白质的表达发生改变；而在相同条件下，EA．hy926v1细胞(EA．hy926的转化细胞株)中有另外45个蛋白点的表达发生变化，说明射频电磁场影响了两种细胞中不同蛋白质的表达。Sul等将4种不同组织来源的细胞暴露于2 mT正弦磁场中，每天辐照1、3和6小时，共14天，发现4种细胞对电磁场的反应性不同。生物系统的遗传特性决定了各生物系统对不同频率电磁场的反应敏感性不同，是导致目前许多研究结果不一致的原因之一。因此，我们认为只有以电磁场敏感细胞为研究对象，才能正确揭示电磁场的生物学效应和作用机制。在本博士论文的第二部分，我们利用传统双向电泳技术进行了电磁场相对敏感细胞的筛选，以为今后的深入研究奠定基础。第一部分：应用双向电泳技术研究电磁场对人乳腺癌细胞蛋白质表达的影响流行病学调查显示极低频电磁场暴露可引起乳腺癌发病率增高。在以往研究的基础上，我们选用50 Hz 0.4 mT正弦磁场对人乳腺癌细胞MCF-7进行辐照和假辐照处理24小时，提取总蛋白质进行双向电泳。银染图谱经PDQuest7.1软件分析显示，磁场辐照组中有6个蛋白质斑点的表达量发生显著改变，同时，在磁场辐照组中有19个蛋白点消失和19个蛋白点新出现。3个差异表达的蛋白质斑点经LC-ESI-IT串联质谱分析，鉴定为RNA结合蛋白调节亚基、蛋白酶体β亚基7型前体和翻译调控肿瘤蛋白。为系统研究射频电磁场对MCF-7细胞蛋白质表达的影响，选择不同时间(1、3、6、12和24小时)、不同强度(SAR为2或3.5 W／kg)、不同辐照模式(5 min-on／10 min-off或连续辐照)的217 Hz调制的全球移动通讯系统(GSM)1800 MHz射频电磁场辐照细胞，然后提取总蛋白质进行双向电泳。结果显示，在本实验条件下，1800 MHz射频电磁场对MCF-7细胞蛋白质表达谱有一定影响，但不明显，且依赖于电磁场暴露的强度、时间和模式。在上述基础上，选择作用较为明显的实验参数(SAR为3.5 W／kg，间断辐照3小时)对MCF-7细胞进行辐照，提取总蛋白质进行荧光差异双向电泳(DIGE)。采用“Decyder”软件进行分析，发现5个蛋白质点表达受电磁场作用上调。三个蛋白经MALDI-TOF／TOF质谱鉴定为CLIC1蛋白、翻译调控肿瘤蛋白和硫醇特异性抗氧化蛋白。另外两个蛋白未得到鉴定。第二部分：应用双向电泳技术筛选电磁场敏感细胞选用来源于不同物种或组织的细胞，包括中国仓鼠肺成纤维细胞CHL、小鼠胚胎成纤维细胞NIH3T3、大鼠肾上腺嗜铬细胞PC12、人眼晶状体上皮细胞SRA01／04、人羊膜上皮细胞FL、人早幼粒白血病细胞HL60和人皮肤成纤维细胞HSF分别暴露于0.4 mT的50 Hz磁场24小时或SAR为3.5 W／kg的1800 MHz射频电磁场间断辐照3小时后，立即提取全蛋白，进行双向电泳。结果显示，工频磁场辐照后，PC12和FL细胞中分别检测到差异表达蛋白点共14个和23个，分别占总检测蛋白点数2.2％和3.2％，而在其余细胞中仅检测到小于1.4％的蛋白质表达发生变化；射频电磁场辐照后，NIH3T3、FL和HL60细胞中分别检测到表达差异蛋白点共20个、23个和17个，分别占总检测蛋白点数2.4％、3.5％和2.0％，在其余细胞中仅检测到小于1.3％的蛋白质表达发生变化。根据检测到的差异点数量及其占总检测蛋白质点数的百分比，结合第一部分结果，初步认为在本实验条件下，MCF-7、PC12和FL细胞为工频磁场的相对敏感细胞，NIH3T3、FL和HL60细胞为射频电磁场的相对敏感细胞。结论： 1．0.4 mT50 Hz磁场可诱导人乳腺癌细胞MCF-7蛋白质表达谱发生显著改变。已鉴定的三个差异蛋白和细胞骨架结构存在一定联系，提示细胞骨架很可能是电磁场作用的靶标。 2．1800 Mnz射频电磁场处理并不能显著改变MCF-7细胞的蛋白质表达模式，提示MCF-7细胞对较高频率的射频电磁场反应性较弱。同时，该弱作用受电磁场辐照强度、作用时间和作用模式等参数的影响。 3．细胞遗传和／或表观遗传(epigenetic)背景决定了其对电磁场的敏感性。在本实验条件下，MCF-7、PC12和FL细胞为工频磁场的相对敏感细胞，NIH3T3、FL和HL60细胞为射频电磁场的相对敏感细胞。不同细胞对电磁场的敏感性不同，同种细胞对不同频段的电磁场反应也可以不一样。 4．蛋白质组学技术可以应用于电磁场生物学效应及机制研究。但由于环境低强度电磁场是一种弱作用因素，易受外界其它因素和细胞本身状态的影响；而蛋白质组学这种高通量技术本身是以牺牲敏感性为代价的，在应用于低强度电磁场这种弱效应研究的过程中，还存在一定的不足。因此，一方面需探索发展更灵敏、更高通量的技术；另一方面需通过国际合作，探索建立蛋白质组学技术在电磁场生物学效应研究中应用的技术标准和规范。从目前的情况看，由于蛋白质学技术本身存在的局限性，对所获得的结果还需谨慎对待，并应通过低通量的常规方法验证。 5．通过对传统双向电泳技术与DIGE技术的比较，我们认为DIGE技术在电磁场应用中并不比传统双向电泳具有更大的优势。本博士论文的创新点： 1．在国际上率先采用蛋白质组学技术进行了电磁场对人乳腺癌细胞蛋白质表达影响的研究及电磁场敏感细胞的筛选工作，在技术手段上有所创新。 2．首次在国际上报道0.4 mT 50 Hz磁场可诱导人乳腺癌细胞MCF-7蛋白质表达谱发生显著改变，并鉴定了3个差异蛋白。 3．首次从蛋白质组学的角度证明MCF-7细胞对1800 MHz射频电磁场的反应性较弱。 4．在国际上首次利用蛋白质组学技术筛选了电磁场的敏感细胞，确定在本实验条件下，MCF-7细胞、PC12细胞和FL细胞为工频磁场的相对敏感细胞；NIH3T3细胞、FL细胞和HL60细胞为射频电磁场的相对敏感细胞。 5．通过对两类电磁场的平行研究，证明不同细胞对电磁场的敏感性不同，而同一种细胞对不同电磁场的反应不同。
[Abstract]:With the rapid development of wireless communication technology and electric power industry, electromagnetic radiation has become one of the fastest growing and most common factors in the environment. The awareness and prevention of its health hazards are related to the sustainable development of science and technology, economy and society in China. An epidemiological survey shows that exposure to ELF EMF can cause leukemia. The incidence of breast cancer and other diseases increases; radio frequency electromagnetic field (RF EMF) exposure of mobile phones can affect the function of the central nervous system and lead to malignant lesions such as brain tumors. These epidemiological findings promote the study of the biological effects and mechanisms of the electromagnetic field on the organism. In vivo, in vitro experimental research suggests that low intensity electromagnetic fields are to God. Through the system, the reproductive system and the immune system can have a certain effect, but there are also a large number of negative reports that cause no correct assessment of the health risk of the electromagnetic field. The cause of this is the original physical process of the electromagnetic field and the biological action, the biological reaction caused and the biological effects. There is a certain blindness in the study of the biological effects of electromagnetic fields. Therefore, it is an urgent problem to be solved to reveal the biological effects and mechanism of the low intensity electromagnetic field.
Various physiological and biochemical changes produced by the electromagnetic radiation of the biological system may involve the regulation of gene expression. Some studies have found that electromagnetic fields can change the mRNA level of proto oncogene, apoptosis related gene, cyclical regulation gene and so on, such as the extremely low frequency electromagnetic field can induce the transcription of the proto oncogene c-myc, c-jun and c-fos, and change the mouse embryonic stem cells Apoptosis related genes Bcl-2 and Bax, cell cycle regulation related gene GADD45 transcription: a certain intensity radio frequency electromagnetic field can down regulate the expression of neuron specific Nurrl gene and up regulate the level of Bax, GADD45 mRNA; low frequency electromagnetic field intermittent irradiation can increase the level of c-jun, p21 and Egr-1 mRNA in p53 deficient cells, but wild type cells are not affected Effect: 1710 MHz radio frequency electromagnetic field can significantly increase the transcription of Hsp70 mRNA in p53 deficient cells, and make c-Jun, c-myc and p21 mRNA increase at a low speed.
The function of the gene in the organism is embodied at the level of the protein at the end of the cell, so the law of life activity can be revealed from the point of view of the protein. The effect of electromagnetic field on the expression of cell protein is not much, mainly focused on the guanosine decarboxylase ODC, the heat shock protein HSP27 / 70 and some signal transduction The influence of the posttranslational modifications, such as the expression levels of PKA, PKC, TPK, MAPK, or phosphorylation of the signal molecules. However, in these studies, the experimenters usually choose the target and mechanism according to the possible effect of the effect of the electromagnetic field, and choose the related single or several proteins to detect. This research idea is based on hypothesis. It may produce subjective deviations, and at the same time, because the selected indexes are scattered and cannot be systematical and integral, they can not fully reveal the biological effects of the electromagnetic field and draw out their reaction pathways. Generally, the electromagnetic field, as a low energy environmental factor, may act on a complex signal transmission process. It is necessary to study the biological effects of the electromagnetic field from the point of view of the proteome. The proteomics method using two dimensional electrophoresis as a separation technique and a mass spectrometry as an identification technique can simultaneously separate hundreds of different kinds of cells. Protein, and compared the changes in protein expression in different physiological or pathological conditions, provides a new method to reveal the influence of external factors on organisms and the pathogenesis of disease.2001. This technology is first introduced by our laboratory and the Finland Leszczynski research team to study the biological effects and mechanisms of electromagnetic fields. In order to explore the possible promotion effect of electromagnetic field on the occurrence of tumor and the similarities and differences of the most common two kinds of electromagnetic fields in the comparative environment, the first part of this thesis selected human breast cancer cell line MCF-7, and studied the effect of 50 Hz extremely low frequency magnetic field and 1800 MHz radio frequency electromagnetic field on the expression of protein in human breast cancer cell line (2-DE). A differential expression map of electromagnetic fields in the cell was established, and some electromagnetic field reaction proteins were identified by mass spectrometry (MS).
According to the results of the first part of the study and the reports in some literature, we think it is necessary to screen and determine the sensitive cells of the electromagnetic field, and to this end, we have set up a further second part of the study. It is generally believed that the biological effects of electromagnetic fields on the cell are affected by the multiple factors of the electromagnetic field itself, such as electromagnetic frequency, intensity, exposure time and mode. However, relative to the influence of the electromagnetic field itself, the source of the biological system (cell / tissue, etc.) and the specific state of irradiation can affect the final result of the experiment.Leszczynski and so on. The expression of 38 proteins can be changed by irradiating EA.hv926 cells of the GSM 900 field of 2.4 W / kg, and the expression of 38 proteins can be changed in 1 hours. Under the same condition, the expression of 45 other protein spots in EA.hy926v1 cells (EA.hy926 transformed cell line) showed that radio frequency electromagnetic fields affected the expression of different proteins in two cells,.Sul and other cells exposed to 4 different tissue sources in the 2 mT sinusoidal magnetic field, irradiated for 1,3 and 6 hours a day for 14 days, and 4 cells were found. The response to electromagnetic fields is different. The genetic characteristics of biological systems determine that the sensitivity of various biological systems to different frequencies of electromagnetic fields is different, which is one of the reasons why many research results are inconsistent. Therefore, we think that the biological effects of electromagnetic fields can be correctly revealed only by using electromagnetic field sensitive cells as the study of the image. In the second part of this doctoral thesis, we use the traditional two-dimensional electrophoresis technology to screen the relative sensitive cells of the electromagnetic field, which will lay the foundation for further research.
Part I: two dimensional electrophoresis was used to study the effect of electromagnetic fields on protein expression in human breast cancer cells.
The epidemiological investigation showed that the exposure of the extremely low frequency electromagnetic field could cause the increase of the incidence of breast cancer. On the basis of previous studies, we used 50 Hz 0.4 mT sinusoidal magnetic field to irradiate and irradiate the human breast cancer cell MCF-7 for 24 hours, and extract the total protein for two-dimensional electrophoresis. The silver staining atlas was analyzed by PDQuest7.1 software, and the magnetic field was shown. The expression of 6 protein spots in the irradiated group changed significantly. At the same time, 19 protein spots disappeared in the magnetic field irradiation group and the protein spots of the 19 protein spots appeared on the 19 protein spots. The protein spots were analyzed by LC-ESI-IT tandem mass spectrometry, which were identified as the RNA binding protein regulating subunit, the proteasome beta subunit 7 precursor and the regulation of the tumor eggs. White.
In order to systematically study the effect of radio frequency electromagnetic field on the protein expression of MCF-7 cells, select different intensities (1,3,6,12 and 24 hours), different intensities (SAR 2 or 3.5 W / kg), 217 Hz modulated global mobile communication system (GSM) 1800 MHz (GSM) 1800 MHz radio-frequency electromagnetic field irradiated by 217 Hz (5 min-on / 10 min-off or continuous irradiation), and then extract the total number of cells. The results showed that the 1800 MHz radiofrequency electromagnetic field had a certain influence on the protein expression profiles of MCF-7 cells in this experimental condition, but it was not obvious and depended on the intensity, time and mode of electromagnetic field exposure. On the basis of the above, the experimental parameters (SAR 3.5 W / kg, intermittent irradiation for 3 hours) were selected. MCF-7 cells were irradiated, and total protein was extracted by fluorescence differential two-dimensional electrophoresis (DIGE). Using "Decyder" software, the expression of 5 protein points was up regulated by electromagnetic field. Three proteins were identified as CLIC1 protein through MALDI-TOF / TOF mass spectrometry, and two other protein and thiol specific antioxidant protein were translated and regulated. The protein was not identified.
The second part: two dimensional electrophoresis is used to screen electromagnetic field sensitive cells.
Cells derived from different species or tissue, including Chinese hamster lung fibroblast CHL, mouse embryonic fibroblast NIH3T3, rat adrenal chromaffin cell PC12, human eye lens epithelial cells SRA01 / 04, human amniotic epithelial cells FL, human promyelocytic leukemic cells HL60 and human skin fibroblasts HSF respectively exposed to 0.4 mT 50 Hz magnetic field 24 hours or SAR 3.5 W / kg 1800 MHz radiofrequency electromagnetic field irradiated for 3 hours after 3 hours, the total protein was extracted and two-dimensional electrophoresis. The results showed that after the frequency magnetic field irradiation, there were 14 and 23 differentially expressed proteins in PC12 and FL cells respectively, which accounted for 2.2% and 3.2% of the total detection protein points, respectively, and in the rest of the cells. Only less than 1.4% of protein expression was detected. After radiofrequency electromagnetic radiation, 20, 23 and 17 differentially expressed protein points were detected in NIH3T3, FL and HL60 cells. The total protein points were 2.4%, 3.5% and 2%, respectively. The protein expression of less than 1.3% was detected in the rest of the cells. According to the number of detected difference points and the percentage of the total detected protein points, combined with the results of the first part, it is preliminarily believed that under this experimental condition, MCF-7, PC12 and FL cells are relatively sensitive cells of the frequency magnetic field, and NIH3T3, FL and HL60 cells are relatively sensitive cells of the radio frequency electromagnetic field.
Conclusion:
The 1.0.4 mT50 Hz magnetic field can induce significant changes in MCF-7 protein expression profiles in human breast cancer cells. There is a certain connection between the three differential proteins identified and the cytoskeleton structure, suggesting that the cytoskeleton may be a target for the effect of electromagnetic fields.
2.1800 Mnz radiofrequency electromagnetic field treatment does not significantly alter the protein expression patterns of MCF-7 cells, suggesting that MCF-7 cells have a weak response to a high frequency electromagnetic field, and the weak effect is influenced by the intensity of electromagnetic radiation, the time and mode of action.
3. cell inheritance and / or epigenetic (epigenetic) background determines its sensitivity to electromagnetic fields. Under this experimental condition, MCF-7, PC12 and FL cells are relatively sensitive cells of the frequency magnetic field, NIH3T3, FL and HL60 cells are relatively sensitive cells of the radio frequency electromagnetic field. Different cell cells have different sensitivity to electromagnetic fields, and the same cells are different The electromagnetic field reaction in the frequency band can also be different.
4. proteomics technology can be applied to the study of the biological effects and mechanisms of electromagnetic fields. However, because the low intensity electromagnetic field is a weak factor, it is easily affected by other factors and the state of the cell itself; and the high throughput technology of proteomics is at the expense of sacrificial sensibility and is applied to low intensity electricity. There are still some shortcomings in the study of this weak effect. Therefore, it is necessary to explore the technology of developing more sensitive and higher flux. On the other hand, we need to explore the technical standards and specifications for the application of proteomics technology in the study of the biological effects of electromagnetic fields through international cooperation. The limitations of the technology itself should be treated with caution and should be verified through low flux conventional methods.
5. by comparing traditional two-dimensional electrophoresis with DIGE technology, we think that DIGE technology is not more advantageous than traditional two-dimensional electrophoresis in the application of electromagnetic field.
The innovation of this doctoral thesis:
1. the study on the effect of electromagnetic field on the expression of protein in human breast cancer cells and the screening of electromagnetic sensitive cells were carried out by proteomics technology in the world, and the technical means have been innovated.
2. for the first time, it is reported internationally that 0.4 mT 50 Hz magnetic field can induce MCF-7 protein in human breast cancer cells.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R35

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