本文選題：電磁場(chǎng) + 極低頻電磁場(chǎng)�。� 參考：《浙江大學(xué)》2007年博士論文

【摘要】： 隨著無(wú)線通訊技術(shù)和電力事業(yè)的飛速發(fā)展，電磁輻射已成為環(huán)境中增長(zhǎng)最快、影響最為普遍的因素之一，對(duì)其健康危害的認(rèn)識(shí)和預(yù)防事關(guān)我國(guó)科技、經(jīng)濟(jì)和社會(huì)的可持續(xù)發(fā)展。有流行病學(xué)調(diào)查顯示極低頻電磁場(chǎng)(ELF EMF)暴露可引起白血病和乳腺癌等發(fā)病率增高；移動(dòng)電話的射頻電磁場(chǎng)(RF EMF)暴露可影響中樞神經(jīng)系統(tǒng)功能，導(dǎo)致腦瘤等惡性病變。這些流行病學(xué)調(diào)查結(jié)果推動(dòng)了電磁場(chǎng)對(duì)生物體的生物學(xué)效應(yīng)及其機(jī)理的研究。體內(nèi)、體外的實(shí)驗(yàn)研究提示低強(qiáng)度電磁場(chǎng)對(duì)神經(jīng)系統(tǒng)、生殖系統(tǒng)和免疫系統(tǒng)等可產(chǎn)生一定影響，但同時(shí)也有大量的陰性報(bào)道存在，導(dǎo)致無(wú)法對(duì)電磁場(chǎng)的健康危險(xiǎn)度進(jìn)行正確評(píng)估。造成這種現(xiàn)象的原因在于電磁場(chǎng)與生物體作用的原初物理過(guò)程、引發(fā)的生物學(xué)反應(yīng)以及產(chǎn)生生物學(xué)效應(yīng)的機(jī)制不清，電磁場(chǎng)生物學(xué)效應(yīng)的研究存在一定的盲目性。因此，揭示低強(qiáng)度電磁場(chǎng)生物學(xué)效應(yīng)及作用機(jī)制成為目前迫切需要解決的問(wèn)題。 生物系統(tǒng)受電磁場(chǎng)輻照所產(chǎn)生的各種生理生化改變可能涉及到基因的表達(dá)調(diào)控。有研究發(fā)現(xiàn)，電磁場(chǎng)可改變?cè)┗�、凋亡相關(guān)基因、周期調(diào)控基因等的mRNA水平，如極低頻電磁場(chǎng)可誘導(dǎo)原癌基因c-myc、c-jun和c-fos的轉(zhuǎn)錄，改變鼠胚胎干細(xì)胞凋亡相關(guān)基因bcl-2和bax、細(xì)胞周期調(diào)控相關(guān)基因GADD45的轉(zhuǎn)錄：一定強(qiáng)度射頻電磁場(chǎng)可下調(diào)神經(jīng)元特異性Nurrl基因的表達(dá)，上調(diào)bax、GADD45 mRNA的水平；低頻電磁場(chǎng)間斷輻照可上調(diào)p53缺陷型細(xì)胞中c-jun、p21和egr-1 mRNA的水平，但野生型細(xì)胞不受影響：1710 MHz射頻電磁場(chǎng)可顯著上調(diào)p53缺陷型細(xì)胞中Hsp70 mRNA的轉(zhuǎn)錄，同時(shí)使c-jun、c-myc和p21 mRNA瞬時(shí)低幅度增加。 基因在生物體的功能最終由其編碼的蛋白質(zhì)在細(xì)胞水平上體現(xiàn)，因此從蛋白質(zhì)的角度入手才能真正揭示生命活動(dòng)的規(guī)律。電磁場(chǎng)對(duì)細(xì)胞蛋白質(zhì)表達(dá)的作用研究不多，主要集中在對(duì)鳥苷酸脫羧酶ODC、熱休克蛋白HSP27／70以及一些信號(hào)轉(zhuǎn)導(dǎo)途徑中信號(hào)分子PKA、PKC、TPK、MAPK等表達(dá)水平或磷酸化等翻譯后修飾的影響上。然而在這些研究中，實(shí)驗(yàn)者通常是根據(jù)推測(cè)的電磁場(chǎng)作用的可能效應(yīng)、作用靶點(diǎn)和機(jī)制，選擇相關(guān)的單個(gè)或幾個(gè)蛋白質(zhì)進(jìn)行檢測(cè)。這種研究思路是以假說(shuō)為前提的，可能產(chǎn)生主觀偏差；同時(shí)，由于所選擇的指標(biāo)是零散的，無(wú)法得到系統(tǒng)性、整體性的結(jié)果，不能全面揭示電磁場(chǎng)的生物學(xué)效應(yīng)，勾畫出其反應(yīng)通路。一般認(rèn)為，電磁場(chǎng)作為一種低能量的環(huán)境因素，可能通過(guò)復(fù)雜的信號(hào)傳遞過(guò)程作用于細(xì)胞，改變多個(gè)蛋白質(zhì)的表達(dá)水平和／或翻譯后修飾，進(jìn)而產(chǎn)生一系列后續(xù)效應(yīng)。因此，從蛋白質(zhì)組的角度研究電磁場(chǎng)的生物學(xué)效應(yīng)是必要的。以雙向電泳作為分離技術(shù)和質(zhì)譜作為鑒定技術(shù)的蛋白質(zhì)組學(xué)方法能同時(shí)分離細(xì)胞內(nèi)成百上千種蛋白質(zhì)，并比較不同生理或病理狀態(tài)下蛋白質(zhì)表達(dá)的變化，為揭示外界因素對(duì)生物體的影響和疾病發(fā)生機(jī)制等提供了一種全新的研究方法。2001年，本實(shí)驗(yàn)室和芬蘭Leszczynski研究小組率先將該技術(shù)引入到電磁場(chǎng)生物學(xué)效應(yīng)及機(jī)制的研究中。為探討電磁場(chǎng)對(duì)腫瘤發(fā)生的可能促進(jìn)效應(yīng)和比較環(huán)境中最常見的兩類電磁場(chǎng)作用的異同，本博士論文第一部分選擇人乳腺癌細(xì)胞株MCF-7，，采用雙向電泳技術(shù)(2-DE)研究了50 Hz極低頻磁場(chǎng)和1800 MHz射頻電磁場(chǎng)對(duì)細(xì)胞蛋白質(zhì)表達(dá)的影響，建立了該細(xì)胞的電磁場(chǎng)蛋白質(zhì)差異表達(dá)圖譜，進(jìn)而利用質(zhì)譜技術(shù)(MS)鑒定了部分電磁場(chǎng)反應(yīng)蛋白質(zhì)。 根據(jù)第一部分的研究結(jié)果和一些文獻(xiàn)的報(bào)道，我們認(rèn)為有必要篩選確定電磁場(chǎng)作用敏感細(xì)胞，為此又設(shè)定了第二部分的研究?jī)?nèi)容。一般認(rèn)為，電磁場(chǎng)對(duì)細(xì)胞的生物學(xué)效應(yīng)受電磁場(chǎng)自身多因素的影響，如電磁場(chǎng)頻率、強(qiáng)度、暴露時(shí)間和模式等。然而，相對(duì)于電磁場(chǎng)自身因素的影響而言，生物系統(tǒng)(細(xì)胞／組織等)的來(lái)源和輻照時(shí)的具體狀態(tài)更能影響實(shí)驗(yàn)的最終結(jié)果。Leszczynski等發(fā)現(xiàn)SAR為2.4 W／kg的GSM 900射頻場(chǎng)輻照EA．hv926細(xì)胞1小時(shí)可引起38個(gè)蛋白質(zhì)的表達(dá)發(fā)生改變；而在相同條件下，EA．hy926v1細(xì)胞(EA．hy926的轉(zhuǎn)化細(xì)胞株)中有另外45個(gè)蛋白點(diǎn)的表達(dá)發(fā)生變化，說(shuō)明射頻電磁場(chǎng)影響了兩種細(xì)胞中不同蛋白質(zhì)的表達(dá)。Sul等將4種不同組織來(lái)源的細(xì)胞暴露于2 mT正弦磁場(chǎng)中，每天輻照1、3和6小時(shí)，共14天，發(fā)現(xiàn)4種細(xì)胞對(duì)電磁場(chǎng)的反應(yīng)性不同。生物系統(tǒng)的遺傳特性決定了各生物系統(tǒng)對(duì)不同頻率電磁場(chǎng)的反應(yīng)敏感性不同，是導(dǎo)致目前許多研究結(jié)果不一致的原因之一。因此，我們認(rèn)為只有以電磁場(chǎng)敏感細(xì)胞為研究對(duì)象，才能正確揭示電磁場(chǎng)的生物學(xué)效應(yīng)和作用機(jī)制。在本博士論文的第二部分，我們利用傳統(tǒng)雙向電泳技術(shù)進(jìn)行了電磁場(chǎng)相對(duì)敏感細(xì)胞的篩選，以為今后的深入研究奠定基礎(chǔ)。 第一部分：應(yīng)用雙向電泳技術(shù)研究電磁場(chǎng)對(duì)人乳腺癌細(xì)胞蛋白質(zhì)表達(dá)的影響 流行病學(xué)調(diào)查顯示極低頻電磁場(chǎng)暴露可引起乳腺癌發(fā)病率增高。在以往研究的基礎(chǔ)上，我們選用50 Hz 0.4 mT正弦磁場(chǎng)對(duì)人乳腺癌細(xì)胞MCF-7進(jìn)行輻照和假輻照處理24小時(shí)，提取總蛋白質(zhì)進(jìn)行雙向電泳。銀染圖譜經(jīng)PDQuest7.1軟件分析顯示，磁場(chǎng)輻照組中有6個(gè)蛋白質(zhì)斑點(diǎn)的表達(dá)量發(fā)生顯著改變，同時(shí)，在磁場(chǎng)輻照組中有19個(gè)蛋白點(diǎn)消失和19個(gè)蛋白點(diǎn)新出現(xiàn)。3個(gè)差異表達(dá)的蛋白質(zhì)斑點(diǎn)經(jīng)LC-ESI-IT串聯(lián)質(zhì)譜分析，鑒定為RNA結(jié)合蛋白調(diào)節(jié)亞基、蛋白酶體β亞基7型前體和翻譯調(diào)控腫瘤蛋白。 為系統(tǒng)研究射頻電磁場(chǎng)對(duì)MCF-7細(xì)胞蛋白質(zhì)表達(dá)的影響，選擇不同時(shí)間(1、3、6、12和24小時(shí))、不同強(qiáng)度(SAR為2或3.5 W／kg)、不同輻照模式(5 min-on／10 min-off或連續(xù)輻照)的217 Hz調(diào)制的全球移動(dòng)通訊系統(tǒng)(GSM)1800 MHz射頻電磁場(chǎng)輻照細(xì)胞，然后提取總蛋白質(zhì)進(jìn)行雙向電泳。結(jié)果顯示，在本實(shí)驗(yàn)條件下，1800 MHz射頻電磁場(chǎng)對(duì)MCF-7細(xì)胞蛋白質(zhì)表達(dá)譜有一定影響，但不明顯，且依賴于電磁場(chǎng)暴露的強(qiáng)度、時(shí)間和模式。在上述基礎(chǔ)上，選擇作用較為明顯的實(shí)驗(yàn)參數(shù)(SAR為3.5 W／kg，間斷輻照3小時(shí))對(duì)MCF-7細(xì)胞進(jìn)行輻照，提取總蛋白質(zhì)進(jìn)行熒光差異雙向電泳(DIGE)。采用“Decyder”軟件進(jìn)行分析，發(fā)現(xiàn)5個(gè)蛋白質(zhì)點(diǎn)表達(dá)受電磁場(chǎng)作用上調(diào)。三個(gè)蛋白經(jīng)MALDI-TOF／TOF質(zhì)譜鑒定為CLIC1蛋白、翻譯調(diào)控腫瘤蛋白和硫醇特異性抗氧化蛋白。另外兩個(gè)蛋白未得到鑒定。 第二部分：應(yīng)用雙向電泳技術(shù)篩選電磁場(chǎng)敏感細(xì)胞 選用來(lái)源于不同物種或組織的細(xì)胞，包括中國(guó)倉(cāng)鼠肺成纖維細(xì)胞CHL、小鼠胚胎成纖維細(xì)胞NIH3T3、大鼠腎上腺嗜鉻細(xì)胞PC12、人眼晶狀體上皮細(xì)胞SRA01／04、人羊膜上皮細(xì)胞FL、人早幼粒白血病細(xì)胞HL60和人皮膚成纖維細(xì)胞HSF分別暴露于0.4 mT的50 Hz磁場(chǎng)24小時(shí)或SAR為3.5 W／kg的1800 MHz射頻電磁場(chǎng)間斷輻照3小時(shí)后，立即提取全蛋白，進(jìn)行雙向電泳。結(jié)果顯示，工頻磁場(chǎng)輻照后，PC12和FL細(xì)胞中分別檢測(cè)到差異表達(dá)蛋白點(diǎn)共14個(gè)和23個(gè)，分別占總檢測(cè)蛋白點(diǎn)數(shù)2.2％和3.2％，而在其余細(xì)胞中僅檢測(cè)到小于1.4％的蛋白質(zhì)表達(dá)發(fā)生變化；射頻電磁場(chǎng)輻照后，NIH3T3、FL和HL60細(xì)胞中分別檢測(cè)到表達(dá)差異蛋白點(diǎn)共20個(gè)、23個(gè)和17個(gè)，分別占總檢測(cè)蛋白點(diǎn)數(shù)2.4％、3.5％和2.0％，在其余細(xì)胞中僅檢測(cè)到小于1.3％的蛋白質(zhì)表達(dá)發(fā)生變化。根據(jù)檢測(cè)到的差異點(diǎn)數(shù)量及其占總檢測(cè)蛋白質(zhì)點(diǎn)數(shù)的百分比，結(jié)合第一部分結(jié)果，初步認(rèn)為在本實(shí)驗(yàn)條件下，MCF-7、PC12和FL細(xì)胞為工頻磁場(chǎng)的相對(duì)敏感細(xì)胞，NIH3T3、FL和HL60細(xì)胞為射頻電磁場(chǎng)的相對(duì)敏感細(xì)胞。 結(jié)論： 1．0.4 mT50 Hz磁場(chǎng)可誘導(dǎo)人乳腺癌細(xì)胞MCF-7蛋白質(zhì)表達(dá)譜發(fā)生顯著改變。已鑒定的三個(gè)差異蛋白和細(xì)胞骨架結(jié)構(gòu)存在一定聯(lián)系，提示細(xì)胞骨架很可能是電磁場(chǎng)作用的靶標(biāo)。 2．1800 Mnz射頻電磁場(chǎng)處理并不能顯著改變MCF-7細(xì)胞的蛋白質(zhì)表達(dá)模式，提示MCF-7細(xì)胞對(duì)較高頻率的射頻電磁場(chǎng)反應(yīng)性較弱。同時(shí)，該弱作用受電磁場(chǎng)輻照強(qiáng)度、作用時(shí)間和作用模式等參數(shù)的影響。 3．細(xì)胞遺傳和／或表觀遺傳(epigenetic)背景決定了其對(duì)電磁場(chǎng)的敏感性。在本實(shí)驗(yàn)條件下，MCF-7、PC12和FL細(xì)胞為工頻磁場(chǎng)的相對(duì)敏感細(xì)胞，NIH3T3、FL和HL60細(xì)胞為射頻電磁場(chǎng)的相對(duì)敏感細(xì)胞。不同細(xì)胞對(duì)電磁場(chǎng)的敏感性不同，同種細(xì)胞對(duì)不同頻段的電磁場(chǎng)反應(yīng)也可以不一樣。 4．蛋白質(zhì)組學(xué)技術(shù)可以應(yīng)用于電磁場(chǎng)生物學(xué)效應(yīng)及機(jī)制研究。但由于環(huán)境低強(qiáng)度電磁場(chǎng)是一種弱作用因素，易受外界其它因素和細(xì)胞本身狀態(tài)的影響；而蛋白質(zhì)組學(xué)這種高通量技術(shù)本身是以犧牲敏感性為代價(jià)的，在應(yīng)用于低強(qiáng)度電磁場(chǎng)這種弱效應(yīng)研究的過(guò)程中，還存在一定的不足。因此，一方面需探索發(fā)展更靈敏、更高通量的技術(shù)；另一方面需通過(guò)國(guó)際合作，探索建立蛋白質(zhì)組學(xué)技術(shù)在電磁場(chǎng)生物學(xué)效應(yīng)研究中應(yīng)用的技術(shù)標(biāo)準(zhǔn)和規(guī)范。從目前的情況看，由于蛋白質(zhì)學(xué)技術(shù)本身存在的局限性，對(duì)所獲得的結(jié)果還需謹(jǐn)慎對(duì)待，并應(yīng)通過(guò)低通量的常規(guī)方法驗(yàn)證。 5．通過(guò)對(duì)傳統(tǒng)雙向電泳技術(shù)與DIGE技術(shù)的比較，我們認(rèn)為DIGE技術(shù)在電磁場(chǎng)應(yīng)用中并不比傳統(tǒng)雙向電泳具有更大的優(yōu)勢(shì)。 本博士論文的創(chuàng)新點(diǎn)： 1．在國(guó)際上率先采用蛋白質(zhì)組學(xué)技術(shù)進(jìn)行了電磁場(chǎng)對(duì)人乳腺癌細(xì)胞蛋白質(zhì)表達(dá)影響的研究及電磁場(chǎng)敏感細(xì)胞的篩選工作，在技術(shù)手段上有所創(chuàng)新。 2．首次在國(guó)際上報(bào)道0.4 mT 50 Hz磁場(chǎng)可誘導(dǎo)人乳腺癌細(xì)胞MCF-7蛋白質(zhì)表達(dá)譜發(fā)生顯著改變，并鑒定了3個(gè)差異蛋白。 3．首次從蛋白質(zhì)組學(xué)的角度證明MCF-7細(xì)胞對(duì)1800 MHz射頻電磁場(chǎng)的反應(yīng)性較弱。 4．在國(guó)際上首次利用蛋白質(zhì)組學(xué)技術(shù)篩選了電磁場(chǎng)的敏感細(xì)胞，確定在本實(shí)驗(yàn)條件下，MCF-7細(xì)胞、PC12細(xì)胞和FL細(xì)胞為工頻磁場(chǎng)的相對(duì)敏感細(xì)胞；NIH3T3細(xì)胞、FL細(xì)胞和HL60細(xì)胞為射頻電磁場(chǎng)的相對(duì)敏感細(xì)胞。 5．通過(guò)對(duì)兩類電磁場(chǎng)的平行研究，證明不同細(xì)胞對(duì)電磁場(chǎng)的敏感性不同，而同一種細(xì)胞對(duì)不同電磁場(chǎng)的反應(yīng)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R35

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