摘要
目的:以人神经胶质瘤细胞株(SHG44)为生物模型,研究900MHz微波单独及联合γ射线照射对细胞的生物学效应。
方法:将细胞随机分为对照组、2mW/cm2、4mW/cm2和6mW/cm2微波暴露组(各组记为C,M2,M4和M6)。细胞辐照2h/d,连续照射3d后,联合组再接受5Gy的γ射线照射。共分为单独γ射线组、2mW/cm2+γ射线组、4mW/cm2+γ射线组和6mW/cm2+γ射线组(记为I,I+M2,I+M4和I+M6)。γ射线(由苏州大学辐照中心提供)采用60Co辐照,照射剂量5Gy,剂量率为1Gy/min。(1)采用MTT法和克隆法测定细胞的生长增殖情况。(2)利用流式细胞仪检测细胞的周期和凋亡情况。(3)测定各组细胞超氧化物歧化酶(superoxide dismutase,SOD)活力和丙二醛(malondialdehyde,MDA)的含量。(4)用RT-PCR方法和western blot法检测各组细胞中HSP70在mRNA和蛋白水平的表达。(5)以正常大鼠神经胶质细胞为研究对象,选取部分上述指标(MTT法和凋亡)进行检测。(6)用BPCL微弱发光测量仪检测细胞在各生长阶段,微波处理后超微弱发光的变化。
结果:(1)单独微波辐射中,M4和M6组的细胞增殖活性显著下降。联合照射组中,I+M6组细胞的增殖活性与单独γ射线组相比显著下降。各单独微波组细胞的克隆形成率均下降。联合照射组中,与I组相比,I+M4与I+M6组的克隆率显著下降。微波和γ射线对增殖活性和克隆率有协同抑制作用。(2)与单独微波组相比,联合组G1期百分比显著升高,而G2期和S期比例均有所下降,但无统计学差异。单独微波辐射后,细胞凋亡率随着微波强度的增加有上升的趋势且呈直线相关,但各组间无统计学差异。与I组相比,I+M4和I+M6组的凋亡率显著升高。与单独微波组相比,各联合组凋亡率显著上升。微波与γ射线对细胞凋亡率存在协同促进作用。(3)单独微波组中,M6组的MDA含量上升,各单独微波组SOD活性也有上升趋势,但各组间没有统计学差异。与I组相比,各联合组MDA含量均显著上升,I+M6组的SOD活性显著下降。微波与γ射线对MDA含量有协同促进作用,对SOD活性的抑制有加强作用。(4)单独微波照射后,HSP70的表达有上升的趋势,但各组间无统计学差异。γ射线照射后HSP70表达显著下降。微波和γ射线对细胞HSP70的表达无交互作用。(5)大鼠原代神经胶质细胞的增殖活性经微波、γ射线和联合照射处理后,与对照组相比均没有统计学差异,仅γ射线组和联合组有下降的趋势。各处理组均无明显凋亡发生。γ射线使ATP酶的活性显著升高,微波和γ射线对ATP酶活性无交互作用。(6)细胞的超微弱发光在接种后第1d即显著增强,一直维持到第5d后显著下降。微波辐射后第1d发光强于对照组,第2d恢复到对照水平。H_2O_2处理后细胞超微弱发光显著增强。
结论:(1)微波和γ射线均能抑制人神经胶质瘤细胞的增殖,且两者之间有协同抑制作用。微波和γ射线还可对细胞氧化损伤产生协同和加强的作用。(2)联合照射未明显抑制大鼠原代神经胶质细胞的增殖和促进其凋亡的发生,但可使ATP酶活性增高。(3)细胞增殖和氧化剂处理能使细胞的超微弱发光明显增强,微波引起细胞发光增强为早期一过性的。
Objective: To study biological effects on SHG44 cells combined by 900MHz microwave andγ-ray radiation.
Methods: SHG44 cells were divided into four groups according to the power density of microwave(0 mw/cm2,2mw/cm2,4mw/cm2 and 6mw/cm2),named as C,M2,M4 and M6,respectively.The cells were exposed to microwave 2h/d for 3d,and then were irradiated byγ-ray (5Gy),whit four groups named as I,I+M2,I+M4 and I+M6,respectively.(1) Cellular proliferation was measured with MTT test and cloning efficiency.(2) Cell cycle and apoptosis were detected by flow cytometer.(3) SOD activity and MDA level were tested by commercial kits.(4) Expression of HSP70 gene was evaluated by RT-PCR and western blot.(5) Normal rat glial cells were cultured and cellular proliferation , apoptosis and ATPase activity were measured.(6) Ultraweak luminescence of cells was measured by the Ultraweak Luminescence Analyzer.
Results: (1) Cell proliferation activity in Group M4 and M6 and cloning efficiency in all exposed groups were decreased by the microwave radiation,with a significant decrease in Group I+M4 and I+M6.Combination of Microwave andγ-ray showed synergistic effect on both cell proliferation activity and the cloning efficiency.(2) Compared to the microwave radiation groups,the percentage of G1 phase cells obviously increased in the combination groups,while that of G2 and S phase decreased.The rate of apoptosis increased with the does of microwave in the microwave radiation groups and so did the I+M4 and I+M6 Group,indicating a synergistic effect ofγ-ray and microwave.(3) The MDA level increased in Group M6 and in all combination groups,and the SOD activity decreased in Group I+M6.(4) Expression of HSP70 at both mRNA and protein levels increased slightly in the microwave exposure groups,but no interaction ofγ-ray and microwave on HSP70 expression was found.(5) Proliferation of the rat glial cell in Group I and I+M6 decreased slightly,and the ATPase activity was significantly increased.(6) Ultraweak luminescence of the exposed cells was stronger than the control cells on the first day,and returned to normal on the second day.
Conclusion: Microwave andγ-ray could inhibit the proliferation of SHG44 cells with a synergistic effect,but such effect was not observed in primary rat glial cells.Ultraweak luminescence could be strengthened by microwave at early stages of exposure.
引文
1.李巧玲,李琳,郭祀远,等.微波生物效应研究的现状及应用[J].生命科学.2001, 13(3):126-128.
2.Herman Cember. Ph.D.. Introduction to Health Physics [M]. NewYork.1996, 628-633.
3 . De Salles.Biological effects of microwave and RF.Microwave and Optoelectronics Conference, 1999.SBMO/IEEE MTT-S, APS and LEOS-IMOC'99.International.1999,51-56.
4.刘亚宁.电磁生物效应[M].北京:北京邮电大学出版社,2002.
5 . S.Banik, S.Bandyopadhyay, S.Ganguly.Bioeffects of microwave-a brief review[J]. Bioresource Technology.2003, 87:155–159.
6.Marina Porcelli, Giovanna Cacciapuoti.Non-thermal effects of microwaves on proteins: thermophilic enzymes as model system [J].FEBS Letters, 1997, 402:102-106.
7.Vera G.V, et al.The relationship between colony-forming ability, chromosome aberrations and incidence of micronuclei in V79 Chinese hamster cells exposed to microwave radiation [J].Mutation Research Letters.1991, 263(3):143-149.
8.John E.H.Tattersall, Iain R.Scott.Effects of low intensity radiofrequency electromagnetic fields on electrical activity in rat hippocampal slices [J].Brain Research.2001, 904:43–53.
9.Banik S, Bandopadhyay S, Ganguly S, Dan D. Effect of inoculation of microwave irradiated methanogenic bacteria for improvement of biomethanation from jute caddis.In:Proc. National symposium on frontier studies in microbes and host microbeinteractions [J]. 2002, 2:55–56.
10.Banik S, Bandopadhyay S, Ganguly S,Dan D.Application of microwave technology on methanogenic bacteria for improvement of its efficiency and endavour to increase yield of biogas.Proc.In:Nineth West Bengal State Science and Technology congress.2002,2:18.
11.Jocelyn A L, Peter W F. Biological effects of Electromagnetic Fields: Mechanisms for the effects of pulsed microwave radiation on protein conformation [J].J.theor.Biol.2000, 206:291-298.
12.W.Grundler.Intensiy- and frequency-dependent effects of microwaves on cell growth rates [J]. Bioelectrochemistry and Bioenergetic.1992, 27(3):361-365.
13.Cole, Kohn.Calcium-mediated signal transdation: biology, biochemistry and therapy [J]. Cancer Metasta-sis Rev.1994, 13:33-41.
14.陈树德,张红锋,等.低频电磁场对细胞生物效应的研究[J].Chin J Phys Med..1998, 20(2):78-80.
15.翟建才,蒋洪,马显光.微波辐射场中的薄层材料界面屏蔽和防护作用的实验研究[J].第三军医大学学报,2006,28(18)1876-1880.
16.张美辨.微波的致突变、致癌、致畸作用[J].国外医学卫生学分册. 2000, 27(5):266-268.
17.林克椿.生物物理学[M].武汉:华中师范大学出版社,1999.
18.Matsumura S, Matsumura T, Ozeki S,et al.Comparative analysis of G2 arrest after irradiation with 75 keV carbon-ion beams and 137Cs Gamma-rays in a human lymphoblastoid cell line[J].Cancer Detect Prev,2003,27(3):222-228.
19.黄伟,张瑶珍,周剑锋,等.60Coγ 射线照射对 k562 细胞周期变化的影响.辐射防护. 2007, 27(1):38-41.
20.杨瑞,彭端云,高亚兵,等.微波辐射对大鼠海马神经元的损伤效应及其机制研究.中华物理医学与健康杂志. 2006, 28(10):670-673.
21.Zhao T Y, Zou S P, Knapp PE.Exposure to cell phone radiation up-regulates apoptosis genes in primary cultures of neurons and astrocytes [J]. Neurosci Lett.2007, 412(1):34-38.
22.Panagopoulos D J,Chavdoula E D,Nezis I P,et al.Cell death induced by GSM 900-MHz and DCS 1800-MHz mobile telephony radiation [J].Mutat Res, 2007,626(1/2):69-78.
23.Lagroye I, Poncy J L.The effect of 50 Hz electromagnetic fields on the formation of micronuclei in rodent cell lines exposed to gamma radiation [J].Int J Radiat Biol.1997, 72(2):249-254.
24.刘晓秋,赵文正,赵阿津.γ 射线与电磁辐射联合对白血病细胞的作用研究[J].辐射研究与辐射工艺学报. 2002, 20(1):77-80.
25.贾永峰,王水明,王德文,等.γ 射线和微波复合照射小鼠骨髓细胞凋亡形态学特点及 Bax、Bcl22 蛋白表达定量分析[J].中国体视学与图像分析.2006, 11(1):5-8.
26.任东青,郭鹞,郭国祯,等.电磁脉冲和电离辐射对人肠上皮细胞存活率影响的异同[J].第四军医大学学报.2000, 21(3):289-291.
27 . Syljuasen RG, Krolewski B, Little JB. Molecular events in radiation transformation [J].Radiat Res. 2001, 155(1 Pt 2):215-221.
28.Matsui Y,Tsuchida Y,Keng PC.Effects of p53 mutations on cellular sensitivity to ionizing radiation[J].Am J Clin Oncol,2001,24(5):486-490.
29.Skulachev V P.FEBS Lett.1998, 423(2):275-280.
30.丰慧根,李延兰,徐振平等.低剂量 γ 射线与香烟对胎鼠脑蛋白质及 HSP70的影响.中国公共卫生,2001,17(10)909-910.
31.李子孺,赖声礼,罗绍凯等.微波对 K562/MDR 细胞凋亡及热休克蛋白 70 表达的影响.华南理工大学学报( 自然科学版).2002, 30(8):24-27.
32.孙存普,张建中,段绍瑾.自由基生物学导论.合肥:中国科学技术大学出版社. 1999, 7-15.
33.Yavuz O,Turkozkan N,Bilgihan A,et al.The effect of 2-chloroadenosineon lipid peroxide level during experimental cerebral ischemia-reperfusionin gerbils.Free Radic Biol Med.1997,22:337-341.
34.莫简.医用自由基生物学导论.北京:人民卫生出版社.1998.214-219.
35.赵振高,龚书明,高双斌等.微波对大鼠肝、血清中 MDA 含量及 SOD 活性影响.第四军医大学学报.1999, 20 (7):644.
36.Mosser D D,Caron A W,Bourget L,et al.Role oft he human heat shock protein HSP70 in protection against stress-induced apoptosis [J].Mol Cell Biol,1997,17 (9):5317-5327.
37.Wong HR,mannix RJ,Rusnak JM,et al.AM J Respir Cell Mol Biol,1996,15(6):745-751.
38.孟丽,彭瑞云,高亚兵等.高功率微波辐射后下丘脑神经元凋亡和线粒体膜电位与 Ca2+的变化.中华劳动卫生职业病杂志.2006, 24(12):739-741.
39.Kittel A, Siklos L, Thuroczy G, et a1.Qualitative enzyme histoehemistry and microanalysis reveals changes in ultrastructural distribution of calcium and calcium-activated ATPases after microwave irradiation of the medial habenula. Acta Neuropathol (Berl).1996, 92:362-368.
40.夏世钧,吴中亮.分子毒理学基础.湖北科技出版社.2001.
41.王强,曹兆进,白雪涛.900MHz 微波电磁辐射对原代培养的大鼠脑皮质神经元的谷氨酸 AMPA 受体 2 及胞内钙离子浓度的影响.卫生研究.2005, 34(2):155-157.
42.王丽峰,彭瑞云,胡向军等.HPM 辐射对大鼠脑能量代谢的影响研究.中国辐射卫生.2006, 15(3):269-271.
43.赵亚丽,宋锦萍,杨玉华,等.不同强度微波辐射对小鼠脑组织钙、镁 ATP 酶活性的影响[J].中华航天航空医学杂志,2000,11(2):101-104.
44.Zhao YL, Ma HB, Song JP, et a1.Efects of microwave irradiation on ATPase activity and voltage dependent ion channel of rat hippocampus cell membrane [J].Space Med Med Eng(Beijing).2003,16(1):36-40.
45.Takeda M, Kobayashi M. Biophoton detection as a novel technique for cancer imaging [J].Cancer science. 2004, 95 (8):656-61.
46.Slawinska D, Slawinska J.Low level luminescence from biological objects.In: JG.Burred.Chemi and Bioluminescence [M].New York:Marcel Dekker Inc,1985:495.
47.Gurwitsh AG.Die nature des spezifischen erregers derzellteilung [J].Arch EntwMech Org.1923, 100:11-14.
48.Colli L.Further measurement on the bioluminescence of the seeding[J]. Experiment.1955, 11:479-482.
49.Masaki Kobayashi,Motohiro Takeda,Tomoo Sato,et al.In vivo imaging of spontaneous ultraweak photon emission from a rat’s brain correlated with cerebral energy metabolism and oxidative stress[J].Neuroscience Research,1999,(34):103-13.
50.毛大璋,沈恂,张月敬等.代谢抑制剂对萌发绿豆超弱发光的影响[J].生物物理学报.1988, 4(2):116-20.
51 . Cadenas E.Biological chemiluminescence [J]. Photochemistry and Photobiology. 1984, 40:823-830.
52.Wright JR, Rumbaugh RC, Colby HD, et al.The relationship between chemilumine-scence and lipid peroxidation in rat hepatic microsomes[J].Archives of Biochemistry.Biophysics. 1979,192:344-351.
1.S Kohli H, Noorlind Brage,et al.Childhood leukaemia in areas with different radon levels: a spatial and temporal analysis using GIS.Epidemiol Community Health. 2000,54:822-826.
2.H.-Erich Wichmann, Angelika Schaffrath Rosario, et al. Increased lung cancer risk due to residential radon in a poolde and extended analysis of studies in Germany.Health Physics Jan 2005, 88,No.1.
3.Helene B,Margot T,et al.Indoor Radon and Lung Cancer in France.Epidemiology. 2004,15:709-716.
4.M Steinbuch, CR Weinberg,et al.Indoor residential radon exposure and risk of childhood acute myeloid leukaemia.British Journal of Cancer.1999,81(5): 900-906.
5.National Radiological Protection Board Documents of the NRPB: Vol 1 No 1.London: HM Stationery Office, 1990.
6.Anonymous.Protection against radon-222 at home and at work.Annals of the ICRP.1993, 23(2):1-45.
7.苗超,仲恒高,等.氡及其子体对小鼠血液和骨髓细胞的影响.中国血液流变学杂志.2005, 15(2):173-174.
8.孙静. 氡及子体吸入对 BALB/c 小鼠造血功能影响的研究.中国血液流变学杂志.2005, 15(1):24-27.
9.童建,洪承皎,等.氡及其子体对大鼠外周血淋巴细胞 DNA 的损伤效应. 辐射研究与辐射工艺学报. 2001, 19(3):236-138.
10.吕慧敏,董金婵,等.中华放射医学与防护杂志.2002, 22(5):340-342.
11.Marjan Bilban. et al.chromosome aberrations study of pupils in high radon level elementary school.Health Phys.2001,80(2):157-163.
12.Helman B, Lennart F, Hamid V.Alkalinne single cell gel electrophoresis and human biomonitofing for genotoxicity:a study on subjects with residential exposure to radon.Murat Res.1999,442:121-132.
13.彭瑞云,等电磁辐射对猕猴骨髓中造血细胞损伤效应研究[J].中国医学物理学杂志. 2003, 20(3):183-184.
14.Weaver A, et a1.Measurement of long-term culture initiating cells (LTC-ICs) using limiting dilution: comparison of endpoints and stromal support [J].Exp Hematol.1997, 25(13):1333-1341.
15.邓中荣,等.微波对体外培养的骨髓基质细胞脂质过氧化损伤[J].第四军医大学学报. 2003, 24(9):820-821.
16.R.Van Den Heuvel, et a1.Haemopoietic cell proliferation in murine bone marrow cells exposed to extreme low frequency (ELF) electromagnetic fields [J].Toxieol in vitro.2001,15: 351-355.
17.方真华,等.50Hz 电磁场对小鼠骨髓间充质干细胞增殖的影响.中华物理医学与康复杂志.2004, 26(1):1-4.
18.张泽云.微核制定法作为生物剂量计影响因素的研究辐射防护.1993, 13(1):16-23.
19.Garaj-Vrhovac V, Fucic A, Horvat D.The correlation between the frequency of micronuclei and specific chromosome aberrations in human lymphocytes exposed to microwave radiation in vitro.Mutation Research.1992, 281(3):181-186.
20.Garaj-Vrhovac V, Horvat D, Koren Z.The affect microwave radiation on the cell genome.Mutation Research 1990, 243:87-93.
21.Maes A, Versehaeve L, Arroyo A, De Wagter C, Vercruyssen L. In Vitrocytogenetie effects of 2450 MHz waves on human peripheral blood lymphcytes. 1993, 14(6):495-501.
22.Garaj-Vrhovac V, Horvat D, Koren Z.The relationship between colony-forming ability, chromosome aberrations and incidence of micronuclei in V79 Chinese hamster cells exposed to microwave radiation.Mutation research, 1991, 263:143-149.
23.张天许,潘红,等.职业性微波接触者的外周血淋巴细胞染色体畸变及微核分析.1996.
24 .郝晋 , 等 . 电磁辐射的生物医学效应 [M]. 山西 : 山西电子技术出版社.1997,9-18.
25.Simko M.Stimulation of phagocytosis and free radical production in murine macrophages by 50 Hz electromagnetic fields [J].2001, 80(8):562-566.