低強(qiáng)度微波輻射對晶狀體上皮細(xì)胞和晶狀體的損傷及對相關(guān)調(diào)控基因表達(dá)的影響.pdf

1、第一部分：低強(qiáng)度微波輻射阻斷晶狀體上皮細(xì)胞周期及對相關(guān)調(diào)控基因表達(dá)的影響.目的：檢測不同劑量微波輻射對兔晶狀體上皮細(xì)胞(rabbit lcns epithelial cells,RLECs)增殖活性及細(xì)胞周期的影響,并測定微波輻射后細(xì)胞周期調(diào)控基岡P21<'WAFI>、P27<'Kipl>和c-myc表達(dá)量的改變.初步探討體外培養(yǎng)的兔晶狀體上皮細(xì)胞對微波輻射損傷的耐受劑量,以及輻射損傷的分子機(jī)制.1、功率密度高于0.50 mW/cm<'

2、2>的微波輻射可引起RLECs產(chǎn)生形態(tài)學(xué)改變,并阻斷細(xì)胞增殖周期,使細(xì)胞停滯于G<,0>/G<,1>期,這種損傷對晶狀體的生長發(fā)育及維持其正常生理功能具有潛在的危害性.2、低強(qiáng)度微波輻射阻斷晶狀體上皮細(xì)胞增殖周期可能通過分別調(diào)節(jié)P27<'Kipl>和c-myc這兩個(gè)調(diào)控細(xì)胞周期轉(zhuǎn)換的基因而實(shí)現(xiàn)的,對細(xì)胞周期另一重要的調(diào)控基因P21<'WAFI>的表達(dá)不影響.3、微波輻射對細(xì)胞周期G<,1>/S限制點(diǎn)重要調(diào)控因子P27<'Kipl>的調(diào)節(jié)

3、可能為轉(zhuǎn)錄后調(diào)節(jié),主要通過調(diào)節(jié)P27<'Kipl>轉(zhuǎn)錄后的蛋白水平起作用,而對其mRNA的表達(dá)不影響.第二部分：低強(qiáng)度微波輻射對體外培養(yǎng)晶狀體的損傷及其細(xì)胞內(nèi)信號傳導(dǎo)機(jī)制.觀察低強(qiáng)度微波輻射對體外培養(yǎng)晶狀體的損傷,并檢測微波輻射后不同時(shí)間對細(xì)胞內(nèi)信號傳導(dǎo)因子PKC-α、c-fos和c-jun表達(dá)的影響,進(jìn)一步探討低強(qiáng)度微波輻射對晶狀體的損傷及其分子機(jī)制.1、高于2.00mW/cm<'2>的微波連續(xù)輻射8h,可引起體外培養(yǎng)晶狀體的水化程度

4、增加,并使晶狀體出現(xiàn)明顯的皮質(zhì)混濁.2、當(dāng)微波輻射強(qiáng)度高于1.00mW/cm<'2>時(shí),即可導(dǎo)致晶狀體中可溶性蛋白和不溶性蛋白比例改變,在不溶性蛋白中,以脲溶性蛋白比例的增加最為明顯,這種平衡的改變破壞了晶狀體的透明性和折光性.3、微波輻射可能通過激活PKC-α,將輻射刺激信號傳入細(xì)胞內(nèi),進(jìn)一步引起早期快反應(yīng)基岡c-fos和c-jun的快速表達(dá),誘發(fā)一系列的生物學(xué)反應(yīng),對細(xì)胞和晶狀體產(chǎn)生損傷.【關(guān)鍵詞】微波;晶狀體;上皮細(xì)胞;信號傳導(dǎo);

5、基因表達(dá);PKC-α;c-fos;c-junABSTRACTEffects of low power microwave radiation oncultured rabbit lens, lens epithelial cells andrelated gene expressionDepartment of Ophthalmology, Medical School of Zhejiang UniversitySpeciality

6、OphthalmologyPostgraduateKaijun WangSupervisorProf. Ke Yao Increased applications of electromagnetic fields are of great concern to public health.The character of the natural electromagnetic field has been altered signif

7、icantly due to technological progress and numerous man-made sources such as industry, traffic, medicine,radio, television, microwaves, RTV apparatuses etc. These sources of non-ionizing radiation cause atmospheric pollut

8、ion similar to the pollution from various industrial sources.The lens of the eye is derived from ectoderm and grows throughout life. This transparent organ consists of a single, cuboidal layer of epithelial cells on the

9、anterior surface and elongated, terminally differentiated epithelial cells, or fiber cells, in the interior.The lens epithelial layer is critical for lens physiology, and insults to this layer can play a role in lens pat

10、hology. Many studies have found that damage to the epithelium can be an early event in cataractogenesis. However, no systematic studies have investigated the biological effects of low power microwave radiation on lens ep

11、ithelial cells.It is well recognized that microwaves affect the biological functions of living organisms at both the cellular and molecular levels and can lead to the appearance of genotoxic effects. However, the mechani

12、sms by which electromagnetic fields exert their biological effects remain poorly characterized. Special attention has been given toopaque. There were significant differences of the hydration of lens after 2.00 and 5.00mW

13、/cm2 microwave radiation compared to control groups (P<0.05).Microwave radiation induced precipitation of protein: The occupation of WSP in the sum of lens protein was decreased gradually after 1.00mW/cm2 radiation for 8

14、 hours. There were significant differences of the proportion of WSP between microwave groups and control groups at 1.00, 2.00 and 5.00 mW/cm2(P<0.01). In the figure of WSP SDS-PAGE electrophoresis stained by coomassie bl

15、ue, lighten bands of low molecular weight and increase of high molecular weight bands can be seen in microwave groups compared to control groups.RLECs morphological changes induced by microwave radiation: Morphological c

16、hanges in the lens anterior capsule epithelial cells were analyzed with a phase-contrast microscope and a fluorescence microscope. With the increasing of microwave intensity, the cells gradually changed from normal, well

17、-distributed appearance to slim, disorderd. Cells nuclei appeared condensation of karyoplasms or chromatin in microwave groups compared with control groups, which lens epithelial cells showed regular nuclei and uniform c

18、hromatin distribution within the nuclei under the fluorescence microscope observation.Effect of microwave radiation on signal transduction related gene expression of PKCα, c-fos and c-jun: In order to determine possible

19、alterations in gene expression after microwave radiation, genes involved in the signal transduction, such as PKC-α, c-fos and c-jun, were evaluated using western blot analysis. β-actin was used as a loading control.High

20、protein level of PKC- α was showed in normal lens epithelial cells. But its protein concentration was significantly increased in cell membrance and decreased in cell cytoplasm after microwave radiation compared to contro

21、l groups. There was a significantly increased expression of c-fos and c-jun protein after 2.00 mW/cm2 microwave radiation for 2, 4,6 and 8 hours when compared to controls.ConclusionThis study indicates that low power mic

22、rowave radiation higher than 1.00 mW/cm2 can affect the proportion of WSP in cultured rabbit lens, cause changes of lens hydration and induce lens opacity.Thesignal transduction mechanisms in lens epithelial cellls after

23、 exposure to microwave radiation may related to increasing of PKC-α , c-fos and c-jun expression.Key wordslens epithelial cell; lens; microwave radiation; gene expression; cell cycle;P21 WAFIsignal transduction; P27Kipl;

24、 P21WAFI; c-myc; PKC- α; c-fos ; c-juninvestigating the effects of low power microwave radiation (<10 mW/cm2) on cell growth,cell cycle progression and DNA synthesis. In human lymphocytes, 450MHz fields at around 1.0mW/c

25、m2 affected cAMP-independent protein kinase activity. The exposure of glioma cells or lymphocytes to 27 or 2450MHz fields caused dose dependent effects on proliferation. DNA synthesis was increased in glioma cells and su

26、ppressed in lymphocytes.Cell cycle alterations in CHO cells were induced by a 27MHz exposure. The major alteration was an increase in the number of cells in G0/G1, and a decrease of those in M phase. Cytogenetic damage h

27、as been reported in human lymphocytes from blood samples exposed to a 945MHz field. Very low power pulsed exposure of human amnion cells at 960MHz have recently been reported to induce a decrease in cell growth rate with

28、 increased exposure time.in our previous studies, we have demonstrated that low power microwave radiation can induce irreversible damage to rabbit lens epithelial cells (RLECs) in vivo. The aim of this study was to deter

29、mine the effect of microwave radiation on primary cultured RLECs and lens, and related signal transduction mechanisms.Part ⅠLow power microwave radiation inhibits the proliferation of rabbit lens epithelial cells by upre

30、gulating P27Kipl expression Objective The goal of this study was to examine the effects of low power microwave radiation (<10mW/cm2) on the proliferation of cultured rabbit lens epithelial cells (RLECs).MethodsCell cultu

31、re: The rabbits used in this investigation were handled in compliance with the "Guiding Principles in the Care and Use of Animals" (DHEW Publication, NIH 86-23) and according to the tenets of the ARVO Statement for the U

32、se of Animals in Ophthalmic and Vision Research. 12-week-old rabbits weighing 1 kg to 1.5kg were killed by CO2 inhalation. The entire eye was removed and dipped in 75％ ethanol for 30 seconds, then it was openedfrom the p

33、osterior segment. The dissection procedure was performedwith sterileinstruments under a laminar flow hood. Lenses were dissected carefully by a posteriorapproach and washed three times in phosphate-buffered saline to rem

34、ove attached pigmentsand vitreous. The capsule epithelium was dissected by fine forceps and placed in a 35-mm2 culture dish adhering to the plastic. Some drops of culture medium were applied to theepithelium specimens to

35、 prevent drying, and the dishes were put in a humidified CO2 incubator (5％ CO2, 37℃) for 6 hours to allow firm attachment of the capsules. Another 2 ml of culture medium was then added, and the capsules were left for 3 o

36、r 4 days before thefirst trypsinization and subculturing.The medium, which was changed every 2 days, was modified Eagle's medium (MEM, GibcoBRL) with 10 ％ fetal bovine serum (FBS),supplemented with 2mM L-glutamine, 100U/

37、ml penicillin and 100μtg/ml streptomycin (all from Sigma). Cells used in subsequent experiments were generally from passages two to three.Electromagnetic field exposure: To demonstrate the effect of low intensity microwa

38、ve radiation on RLECs, cells were exposed to continuous microwave radiation with a frequency of 2450MHz and power densities of 0.10, 0.25, 0.50, 1.00, and 2.00 mW/cm2 for 8 hours under controlled temperature conditions (

39、25℃). Sham exposed cells were treated in an identical fashion to the exposed cells except that the power to the antenna was not activated. The exact microwave frequency was calculated using a frequency counter,and the in

40、put power was accurately measured using an electromagnetic field detector (Narda Model 8700, USA). There was less than a 0.6℃difference between the temperature of the culture medium in the exposed and sham cells after 8

41、hours of radiation. In order to detect P21WAFI and P27Kipl expression, cells were treated with 2mW/cm2microwave radiation, collected from each culture dish and used for RT-PCR or stored at -80℃until assayed by western bl

42、ot.Morphologic study: In order to detect the influence of microwave radiation on RLECs directly, treated cells were observed att er 8 hours of treatment with 0.10, 0.25, 0.50, 1.00and 2.00 mW/cm2 microwave radiation usin

43、g a phase-contrast microscope (Nikon, Japan)and HE stain.MTT assay: To examine cell viability, an MTT assay was used. Briefly, RLECs were seeded in 96-well cell culture plates at a density of 1 ×106/ml cells in MEM suppl

44、emented with 10％ fetal bovine serum. After microwave radiation, 20μl MTT solution (5mg/ml,Sigma) was added to each well for an additional 4 hours. Then the MTT solution was removed, and the blue MTT formazan precipitate

45、was dissolved in 150μl DMSO.The optical density (A value) of samples was measured at 490nm using an enzymelinked immunosorbent assay plate reader (Bio-Rad, Hercules, CA).Cell cycle analysis: After treated with different

46、doses of microwave radiation for 8 h, cells were cultured in the medium for another 16 h, then trypsinized and harvested by centrifugation, washed with PBS and fixed in 70％ ice-cold ethanol. 105cells were stained With pr

47、opidium iodide (0.5 ml/L in PBS, containing with 100μg/ml RNase A)and subjected to flow cytometric analysis of DNA content using a Coulter Berkman cytometer of which Mcycle software was used for data analysis.Gel electro

48、phoresis and western blot analysis:After exposed to an 2.00 mW/cm2electromagnetic field for 4, 6 and 8 hours, control and treated RLECs were collected,washed with cold PBS and lysed in buffer containing 20mM Tris-HCI (pH

49、8.0), 150mM NaCl,30μg/mlAprotinin,1mMNa3VO4,0.3％SDS,5mMEDTA,1％deoxycholate, and 1mM PMSF. Aftercentrifugation at 12,000 rpm for 5 min at 4℃, protein concentrations were determined using the Lowry method. The samples (30~

50、50μg of total protein) were boiled for 3 min, separated by SDS-polyacrylamide gel electrophoresis at 150 V and transferred to a polyvinylidene difluoride membrane (Hybond, Amersham). After transfer, membranes were blocke

51、d in TBST containing 5％nonfat dry milk for 1h. Western immunoblotting was carried out using the primary antibodies anti-P21WAFI, anti-P27Kipl, c-myc or anti-β-action (Santa Cruz Biotechnology,Santa Cruz, CA) at 1:500 dil

52、ution. The membranes were then reacted with a horseradish peroxidase-conjugated anti-mouse secondary antibody (Santa Cruz) for 2 hours. Signals were detected with an ECL Plus kit (Amersham) according to the manufacturers

53、 instructions.RNA isolation and semiquantitative RT-PCR: Total cellular RNA was isolated from RLECs using the TRIzol reagent (GibcoBRL) according to the manufacturer's protocol.Samples of RNA (1~2μg of total RNA) were re

54、verse transcribed and amplified with genespecific primers using the ThermoScript RT-PCR System kit (GibcoBRL). Primer sequences used to amplify the target genes were: P21WAFI cDNA, 5'-CAG TGG ACA GCG AGC AGC TG (sense) a

55、nd 5'-TAC AAG ACA GTG ACA GGT CC (antisense), 282bp;P27Kipl cDNA, 5'-GAG GGC AAG TAC GAG TGG CAA (sense) and 5'-CTG CGC ATT GCT CCG CTA ACC (antisense), 238bp; GAPDH cDNA, 5'-ATG GTG AAG GTC GGA GTC AAC G (sense) and 5'-

56、GTT GTC ATG GAT GAC CTT GGC C (antisense), 495bp.Expression of GAPDH was used as the internal standard. Amplification was performed in a thermal cycler at 94℃ for 30 seconds, at 56℃ for 20 seconds and at 72℃ for 30 secon

57、ds with 30 cycles. After PCR, 1μl of PCR product was cloned, and nucleotide sequencing of the amplified products was performed by using a gene analyzer (mega Base 1000,Pharmacia,USA) to conform theidentity of the fragmen

58、t.PCR products were electrophoresized on a 2％ agarose gel and visualized with ethidium bromide. The intensities of the amplified cDNA fragment were estimated using a video-densitometer.Samples without reverse transcripta

59、se were used in the RT-PCR procedure as a negative control.Statistical analysisThe data were analyzed by one-way analysis of variance (ANOVA) and x2, a P<0.05 was assumed to be statistically significant.ResultsMorphologi

60、cal changes induced by microwave radiation: Morphological changes in the RLECs were analyzed with a phase-contrast microscope. Compared with control groups, the cell shape changed from a normal, flat, multiangle appearan

61、ce to a slim, spindle-like one in the 0.50mW/cm2 microwave treated groups. Rounded cells were observed in the 1.00mW/cm2 microwave exposed groups, and floating cells were detected in the 2.00 mW/cm2microwave exposed grou

62、ps. The density of adherent cells was decreased in the 0.50, 1.00and 2.00 mW/cm2 microwave treated groups. There were no significant morphologicalchanges in the 0.10 and 0.25 mW/cm2 groups compared with control groups (d

63、ata not shown).Cell viability decreases after microwave radiation: After treated with microwave radiation, RLECs were assayed for Avalue to detect cell viability and compared to control groups. Cell viability significant

64、ly decreased after 8 hours of treatment with 0.50, 1.00 and 2.00 mW/cm2 microwave radiation (P<0.01), but it was not modified in the 0.10 and 0.25mW/cm2 groups (P>0.05). Microwave radiation inhibited cell viability at po

65、wer densities ranging from 0.50 mW/cm2 to 2.00 mW/cm2. Moreover, the high level (2.00 mW/cm2) ofmicrowave radiation inhibited cell viability prominently, the percent viability of cells was only (65.05±5.08)％ compared to

66、the control groups.Microwave radiation blocks RLECs cell cycle in G0/G1 phase: The effect of microwave radiation on the cell cycle was determined by flow cytometry. After 8 hours of treatment with 0.50, 1.00, and 2.00mW/

67、cm2 microwave radiation, the RLECs were arrested in the G0/G1 phase of the cell cycle. The percentage of RLECs in the G0/G1 phase were 71.95±2.12％ (P<0.05), 75.68±3.35％ (P<0.01) and 82.40±8.68％ (P<0.01) respectively.Wher

68、eas the percentage of cells in the S phase of the cell cycle from the microwave-treated groups was significantly lower than controls (P<0.05, P<0.01). Thus, microwave radiation suppressed lens epithelial cell proliferati

69、on by inhibiting and delaying the G1/S transition of the cell cycle. There was no modification to the cell cycle in the 0.10 and 0.25 mW/cm2microwave treated cells, and no significant changes in the percentage of cells i

70、n the G2/M phase between microwave treated and control groups.Effect of microwave radiation on cell cycle related gene expression of P21WAFI, P27Kipl and c-myc: In order to determine possible alterations in gene expressi

71、on after microwave radiation, genes involved in the cell cycle, such as P21WAFI and P27Kipl, were evaluated in the RLECs using western blot analysis. β-actin was used as a loading control. Both the microwave-treated and

72、the untreated cells exhibited P21WAFI and P27Kipl protein expression.However, there was a significantly increased expression of P27Kipl protein in RLECs after 2.00 mW/cm2 microwave radiation for 4, 6 and 8 hours when com

73、pared to controls. There were no detectable differences in P21 WAFI protein levels between the microwave treated andcontrol groups.To confirm the expression levels of P27Kipl and P21WAFI mRNA, RT-PCR was performed.The se

74、quence data of the PCR products were identical with the sequences found in GenBank (Data not shown). Levels of individual PCR products were expressed as the ratio of individual product optical density to that of the inte

75、rnal standard GAPDH. As shown in Figure 5A, the results of RT-PCR analyses showed that there were no detectable differences of P27Kipl mRNA expression in microwave treated groups after 4, 6 and 8 hours of radiationcompar

76、ed with controls (P>0.05). The expression levels of P21WAFI mRNA were also similar in all six groups which correlated with the western blot data. This result suggests that microwave radiation cannot affect the levels of

77、P27Kipl and P21 WAFI mRNA expression.ConclusionThis study suggests that low power microwave radiation higher than 0.50 mW/cm2 can inhibit lens epithelial cell proliferation, increase the expression of P27Kipl, and decrea

78、se the expression of c-myc. These effects may account for the decline of lens epithelial proliferation after exposure to microwave radiation.Part ⅡEffects of microwave exposure on cultured rabbit lens and related signal

79、transduetion mechanismsObjectiveThe goal of this study was to examine the effects of low power microwave radiation (<10mW/cm2) on the cultured rabbit lens, and to try to explore the mechanisms of microwave radiation rela

80、ted signal transduction.MethodsLens organ culture: Rabbits were killed and the eyes were removed. The lenses were carefully dissected by a posterior approach. Each of the dissected lenses was placed in a well of a 6-well

81、 culture plate containing 3.0 ml modified Eagle's medium (MEM) and 10％fetal bovine serum for 6h. Transparent lenses (without surgical damage) were selected and divided into 2 groups randomly for experimentation.Electroma

82、gnetic field exposure: Cultured lenses were exposed to continuous microwave radiation with a frequency of 2450MHz and power densities of 0.25, 0.50, 1.00, 2.00 and 5.00 mW/cm2 for 8 hours under controlled temperature con

83、ditions (25 ℃ ). Sham exposed lenses were treated in an identical fashion to the exposed lenses except that the power to the antenna was not activated. The exact microwave frequency was calculated using a frequency count

84、er, and the input power was accurately measured using an electromagnetic field detector (Narda Model 8700, USA).Lens morphologic study and hydration measurement: After 8 hours of microwave radiation treatment, the variat

85、ion of lens opacity was observed, then washed with PBS and weighted (wi). After that, lenses were placed in a baking oven (100℃) for 16 hours and weighted again (w2). The hydration of lens was the ratio of (w 1-w2) to wi

86、.Analysis of WSP: In order to detect the influence of microwave radiation on lens protein,lenses of water soluble protein (WSP), urea soluble protein (USP), alkali-soluble protein (ASP) and sonicated protein (SP) were st

87、udied using the Lowry method, bovine serum albumin was used as the standard. Simultaneously, WSP was detected by SDS-PAGE electrophoresis stained by coomassie blue.Morphologic study of lens epithelial cells: After washed

88、 with PBS, the lens capsule epithelial cell samples was spread out on a slide and observed using a phase-contrast microscope. In order to detect the influence of microwave radiation on lens epithelial cells nuclei direct

89、ly, a Hoechst33258 staining was used. Briefly, the cells were fixed in 4℃ for 5min, stained by Hoechst33258 for 10 min and washed by ddH2O2 for 3 times, then was observed under a fluorescence microscope.Gel electrophores

90、is and western blot analysis:(1) Extraction of tissues protein: After exposed to 2.00 mW/cm2 electromagnetic field for 2, 4, 6 and 8 hours, control and treated lenses were collected, washed with cold PBS. The lens anteri

91、or capsule was dissected and lysed in buffer containing 20mM Tris-HCI(pH8.0), 150mM NaCl, 301μg/ml Aprotinin, 1mM Na3VO4, 0.3％ SDS, 5mM EDTA,1％ deoxycholate, and 1mM PMSF. Aftercentrifugation at 12,000 rpm for 5 min at 4

92、 ℃, protein concentrations were determined using the Lowry method. The samples was stored in -70℃ before ananlysis by gel electrophoresis.(2) Extraction of membrance protein and cytoplasmic protein: After 2.00 mW/cm2elec

93、tromagnetic field exposure for 2, 4, 6 and 8 hours, the lens was washed, anterior capsule was dissected and lysed in buffer containing 10mmol/l Tris-HCI, 320 mmol/L saccharose (PH7.4). After centrifugation at 700 rpm for

94、 5 min at 4℃, and 37,000 rpm for 30min at 4℃, the supernatant was the cytoplasmic protein, and the sediment dissoved in 10mmol/LTris-HCI(PH7.4)wasmembranceprotein.Proteinconcentrationswere determined using the Lowry meth

95、od, and the samples was stored in -70℃ before ananlysis by gel electrophoresis.The samples (30~50μg of protein) were boiled for 3 min, separated by SDSpolyacrylamide gel electrophoresis at 150 V and transferred to a poly

96、vinylidene difluoride membrane (Hybond, Amersham). After transfer, membranes were blocked in TBSTcontaining 5％ nonfat dry milk for 1h. Western immunoblotting was carried out using the primary antibodies anti-PKC- α , ant

97、i-c-fos, c-jun or anti-β-action (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500 dilution. The membranes were then reacted with a horseradish peroxidase-conjugated anti-mouse secondary antibody (Santa Cruz) for 2 hour

98、s.Signals were detected with an ECL Plus kit (Amersham) according to the manufacturers instructions.Statistical analysisThe data were analyzed by one-way analysis of variance (ANOVA), a P<0.05 was assumed to be statistic

99、ally significant.ResultsLens opacity and hydration changes induced by microwave radiation:Opacification of lens was increased obviously after 5.00mW/cm2 microwave radiation for 8hours. Several vesicles occurred at the eq