高功率微波對視網(wǎng)膜神經(jīng)節(jié)細(xì)胞脂質(zhì)過氧化作用的實(shí)驗(yàn)研究

1、    高功率微波對視網(wǎng)膜神經(jīng)節(jié)細(xì)胞脂質(zhì) 過氧化作用的實(shí)驗(yàn)研究        摘要：目的觀察微波對體外培養(yǎng)的兔視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的脂質(zhì)過氧化損傷作用。方法體外培養(yǎng)兔神經(jīng)節(jié)細(xì)胞，以平均功率為80mW/cm2的微波進(jìn)行輻照，輻照時(shí)間分別為15、30、45min。輻照后立即于倒置顯微鏡和電鏡下觀察細(xì)胞形態(tài)變化，羥胺法和改良硫代巴比妥酸熒光法分別測定細(xì)胞內(nèi)超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)含量。

2、結(jié)果微波輻照后，細(xì)胞軸突消失，電鏡可見線粒體及內(nèi)質(zhì)網(wǎng)腫脹。隨輻照時(shí)間延長，細(xì)胞逐漸趨向于壞死，細(xì)胞MDA含量增高，45min輻照組MDA含量為對照組的5.11倍。SOD則逐漸降低。結(jié)論微波可引起視網(wǎng)膜神經(jīng)節(jié)細(xì)胞脂質(zhì)過氧化損傷，脂質(zhì)過氧化反應(yīng)可能是微波視網(wǎng)膜損傷的作用機(jī)制之一。關(guān)鍵詞：微波/副作用；視網(wǎng)膜神經(jīng)節(jié)細(xì)胞病理學(xué)；脂質(zhì)過氧化作用分類號：R774R538.9文獻(xiàn)標(biāo)識碼：A文章編號：1005-1015（2000）01-0032-03Study of lipid peroxide damage in retinal ganglion cells induced by high power mi

3、crowaveYANG RuihuaCHEN JingyuanHUANG Wenhua（Department of Military Hygiene,the Fourth Military Medical University,Xian 710032，China）Abstract：ObjectiveTo determine the lipid peroxide damage in the primary cultured rabbit retinal ganglion cells induced by microwaveMethodsCultured rabbit retinal gangli

4、on cells in vitro and exposed to 80 mW/cm2 of microwave for 15,30,45 min tespectivelyImmediately after radiation,the morphological variation of cells was observed by optical microscope and transmission electronic microscopeSecondly,the activity of intracellular superoxide dismutase (SOD) and the con

5、tent of malondialdehyde (MDA) were detectedResultsA portion of cells congregated,with their axon disapeared after radiationMitochondria and endoplasmic reticulum revealed swelling under transmission electronic microscopeThe content of MDA was increased obviously compared with control group while SOD

6、 decreasedThe content of MDA as increased obviously compared with control group after 45 min radiation was 511 times,while SOD decreasedThe content of MDA as in control and the ganglion cells were apparantly destroyedConclusionMicrowave can induce the lipid peroxide damage in primary cultured retina

7、l ganglion cells,and lipid peroxide effect might be one of the mechanisms of microwave retinal damageKey Words：Microwaveadvevse effects；Retinal ganglion cellspathology；Lipid peroxidation研究表明，微波可對眼造成明顯損害1-3，導(dǎo)致白內(nèi)障，損傷角膜、虹膜等，微波對視網(wǎng)膜損傷的研究較少。為此，我們觀察了微波對體外培養(yǎng)的兔視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的脂質(zhì)過氧化損傷，以便為進(jìn)一步研究微波的眼底損傷及防護(hù)提供一定的實(shí)驗(yàn)依據(jù)。1材料

8、和方法細(xì)胞培養(yǎng)：家兔由本校中心實(shí)驗(yàn)室提供，6只，黑色或灰色，雄性，體重(1.8±0.68)kg。靜脈氣栓處死家兔，取出眼球，剔除周圍軟組織，75%酒精浸洗，沿眼球赤道環(huán)形剪開鞏膜，由切口處向視盤剝?nèi)∫暰W(wǎng)膜，至視盤處剝離，0.25%胰酶消化，含10%小牛血清的改良依格培養(yǎng)液(Dulbecco?s modified Eagle?s medium,DMEM)終止消化，1000r/min離心5min后棄上清，D-Hank?s液洗滌(1000轉(zhuǎn)，5min)2次，接種于含10%小牛血清的DMEM培養(yǎng)液中，37、5%CO2孵箱培養(yǎng)。細(xì)胞輻照：速調(diào)管微波源，頻率2.856×109 Hz，脈

9、沖波，脈寬2s，平均功率80 mW/cm2，培養(yǎng)72h的細(xì)胞，培養(yǎng)瓶細(xì)胞生長面面向波源，于屏蔽室內(nèi)進(jìn)行輻照。輻照時(shí)間分別為0、10、30、45min。室溫19?20。觀測內(nèi)容：細(xì)胞形態(tài)觀察：輻照后立即于光鏡下觀察細(xì)胞，以胰酶消化收集細(xì)胞，0.3%戊二醛固定后進(jìn)行電鏡觀察。超氧化物歧化酶(SOD)及丙二醛(MDA)測定：胰酶消化收集細(xì)胞，平衡鹽溶液漂洗2次，調(diào)整細(xì)胞數(shù)為1×106/ml，超聲粉碎，取100l細(xì)胞粉碎液分別用羥胺法4和改良硫代巴比妥酸熒光法5測定SOD和MDA。統(tǒng)計(jì)學(xué)處理：用SPSS統(tǒng)計(jì)軟件進(jìn)行t檢驗(yàn)。2結(jié)果細(xì)胞鑒定：細(xì)胞培養(yǎng)35d后，細(xì)胞貼壁，可見到細(xì)胞有軸突伸展(1)

10、?？股窠?jīng)元烯醇化酶(neuron specific enolase,NSE)免疫組織化學(xué)染色陽性，確認(rèn)為神經(jīng)節(jié)細(xì)胞。1正常RGCs光鏡像。細(xì)胞較小，可見軸突伸展活細(xì)胞×250Fig1Normal RGCs under optical microscopeRGCs were small,axons can be seen×250微波輻照對視網(wǎng)膜神經(jīng)節(jié)細(xì)胞形態(tài)的影響：微波輻照后，15min組細(xì)胞軸突消失(2)，電鏡下可見細(xì)胞線粒體腫脹，嵴減少(3)，30min組細(xì)胞發(fā)生聚集，邊緣模糊，電鏡觀察可見凋亡細(xì)胞(4)，60 min組部分細(xì)胞脫壁，可見細(xì)胞碎片，細(xì)胞數(shù)量明顯減少。2微波

11、輻照15min后RGCs光鏡像。RGCs軸突消失活細(xì)胞×250Fig2Configuration of RGCs after 15 min microwave radiation in optical microscopeAxons of RGCs dispeared×2503微波輻照15min后RGCs電鏡像。電鏡下可見細(xì)胞線粒體和內(nèi)質(zhì)網(wǎng)腫脹醋酸鉛-枸櫞酸鉛雙染色×5000Fig3Configuration of RGCs after 15 min microwave radiation detected bytransmission electronic mi

12、croscopeMitochondria and endoplasmic reticulum looked swellingAceticleadandcitricleaddyeing×50004微波輻照30min后RGCs電鏡像。RGCs染色質(zhì)濃縮，邊集為凋亡細(xì)胞醋酸鉛-枸櫞酸鉛雙染色×5000Fig4Configuration of RGCs after 30 min microwave radiation detected by transmission electronic microscopeThe apoptosis RGCs chromosome collect

13、ed and encircled on cell nuclear membrane×5000微波輻照對視網(wǎng)膜神經(jīng)節(jié)細(xì)胞SOD與MDA含量的影響：微波輻照后，MDA顯著升高，且升高輻度與輻照時(shí)間成正相關(guān)；SOD活性則隨輻照時(shí)間延長而逐步下降(表1)。表1不同時(shí)間微波輻照后視網(wǎng)膜神經(jīng)節(jié)細(xì)胞SOD、MDA含量(±s,n=6)Tab1Content of SOD and MDA of retinal ganglion cellsafter microwave radiation (±s,n=6)MDA(mol)SOD(U)control group480±255

14、102±01610 min182±808072±005*30 min1843±252*058±001*45 min2455±219*054±003*t-test*P005,*P001 vs control group 3討論微波對眼損傷主要涉及晶狀體、視網(wǎng)膜和角膜，目前研究較多的是對晶狀體的損傷，而有關(guān)視網(wǎng)膜損傷的報(bào)道較少，且多為組織病理學(xué)損傷的觀察1，2。本實(shí)驗(yàn)以兔視網(wǎng)膜神經(jīng)節(jié)細(xì)胞為對象研究高功率微波輻照的損傷作用，結(jié)果表明：脈沖波輻照可導(dǎo)致體外培養(yǎng)的兔視網(wǎng)膜神經(jīng)節(jié)細(xì)胞發(fā)生形態(tài)學(xué)變化，首先表現(xiàn)為軸突的破壞，細(xì)胞發(fā)生聚集，隨

15、輻照時(shí)間的延長，細(xì)胞形態(tài)進(jìn)一步遭到破壞，60min后已不能觀察到完整的細(xì)胞形態(tài)。有關(guān)微波對視網(wǎng)膜損傷的機(jī)制多認(rèn)為是由于微波的熱效應(yīng)引起6。本實(shí)驗(yàn)中各組溫度無明顯變化，但SOD與MDA已明顯改變。可見，微波除了熱效應(yīng)外，可能還具有非熱效應(yīng)，通過脂質(zhì)過氧化引發(fā)細(xì)胞損傷。視網(wǎng)膜的光感受器外段含大量長鏈不飽和脂肪酸，受到過氧化作用后形成脂質(zhì)自由基，并攻擊其他不飽和脂肪酸引起連鎖反應(yīng)。在微波作用下，核膜、盤膜、線粒體膜和內(nèi)質(zhì)網(wǎng)的脂質(zhì)發(fā)生過氧化，導(dǎo)致膜中蛋白質(zhì)酶和磷脂交聯(lián)失活，使膜的流動性和通透性改變而受到不可逆的損傷，嚴(yán)重者導(dǎo)致生物膜溶解和細(xì)胞死亡。MDA是脂質(zhì)過氧化反應(yīng)的主要降解產(chǎn)物，可作為體內(nèi)自由基

16、水平的間接反映指標(biāo)，MDA含量隨輻照時(shí)間延長而增高說明微波對神經(jīng)節(jié)細(xì)胞的損傷涉及脂質(zhì)過氧化反應(yīng)，同時(shí)清除超氧陰離子自由基的SOD隨輻照時(shí)間延長而逐漸下降，與MDA的變化相對應(yīng)，說明輻照時(shí)間越長，脂質(zhì)過氧化損傷越嚴(yán)重。(本文編輯：唐健)作者單位：楊瑞華（710032 西安，第四軍醫(yī)大學(xué)軍隊(duì)衛(wèi)生學(xué)教研室）陳景元（710032 西安，第四軍醫(yī)大學(xué)軍隊(duì)衛(wèi)生學(xué)教研室）劉學(xué)東（710032 西安，第四軍醫(yī)大學(xué)軍隊(duì)衛(wèi)生學(xué)教研室）鄧中榮（710032 西安，第四軍醫(yī)大學(xué)軍隊(duì)衛(wèi)生學(xué)教研室）劉秀紅（710032 西安，第四軍醫(yī)大學(xué)軍隊(duì)衛(wèi)生學(xué)教研室）龔書明（710032 西安，第四軍醫(yī)大學(xué)軍隊(duì)衛(wèi)生學(xué)教研室）黃文華（西北核技術(shù)研究所）劉國治（西北核技術(shù)研究所）參考文獻(xiàn)：1劉導(dǎo)涪，樓蘇生微波輻射及其對眼的影響J眼外傷職業(yè)眼病雜志1984，2：123-128.2Paulsson LERetinal damage experimentally induced by microwave radiation at 55 mW/cm2JActaOphthalmol,1979,57:183-1853Bhatt N,Peyman GA,Khoobehi B,et alMicrow