【作者】 曹毅；

【导师】 童建；

【作者基本信息】 苏州大学 ， 卫生毒理学， 2009， 博士

【摘要】 目的:在细胞和机体水平研究不同剂量微波和γ射线联合照射对神经系统和造血系统的生物学效应以及低剂量微波辐射对γ射线引起的造血损伤的防护效应及机理,初步分析微波辐射对人群神经行为的影响,为探讨微波和γ射线联合作用的分子机制、微波通讯对神经系统的健康影响及低剂量微波对其他有害因素的防护效应提供基础资料。方法:原代培养的骨髓细胞分别接受单独的微波辐射、单独γ射线辐射以及微波和γ射线的联合照射,分析细胞增殖活性变化、细胞周期的变化。昆明种小鼠分别接受单独的微波辐射、单独γ射线辐射以及微波和Υ射线的联合照射,根据动物存活率、存活时间、外周血计数等客观指标的变化,建立能够有效减轻电离辐射危害的微波辐射的动物模型;采用骨髓有核细胞计数、造血干/祖细胞数量和增殖能力检测、骨髓组织病理变化观察,分析微波对电离辐射造血损伤的拮抗效应及其规律。神经胶质瘤细胞分为对照组、单独微波组、单独γ射线组以及联合暴露组。对照组不接受任何照射,单独微波组接受2mW/cm2、4mW/cm2和6mW/cm2微波辐射,联合组在微波照射后再接受5Gyγ射线照射,单独电离组只接受5Gyγ射线照射。分析不同组别细胞的生长增殖情况、细胞周期和凋亡的改变、超氧化物岐化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)含量,分析各组细胞HSP70的mRNA和蛋白质表达情况。以原代培养的大鼠神经胶质细胞为研究对象,测定不同处理后细胞的增殖情况、凋亡情况以及ATP酶的表达。昆明种小鼠分别接受1.5mW/cm2、3mW/cm2、6 mW/cm2和9mW/cm2的900MHz微波辐射,开放场实验和避暗实验测定小鼠学习记忆功能,测定海马组织中HSP70的mRNA表达水平。选择苏州市区内某移动通讯基站周围长期定居人群作为观察组人群,对照组为非移动基站周围定居人群。对基站周围辐射水平进行测量,收集研究对象的基本情况,采用神经行为测试评价系统对基站周围人群及对照人群进行测试。结果:原代骨髓细胞实验结果:12μW/cm2的900MHz微波辐射能显著提高原代骨髓细胞的增殖活性,影响细胞周期,使G1期细胞比例明显减少(p<0.05),增殖期(S+G2/M)细胞比例显著升高。微波与γ射线联合照射对小鼠造血系统影响的动物实验结果:60Coγ射线照射前给予120μW/cm2的900MHz微波辐射能显著提高受照射小鼠的存活率(p<0.05),促进外周血白细胞恢复,提高受照射小鼠的脾脏和胸腺系数,增强γ射线照射后小鼠骨髓有核细胞(bone marrow nucleated cells,BMNCs)增殖活性,促进CFU-GM集落形成,并刺激造血生长因子GM-CSF、IL-3的表达。单独γ射线与复合照射均可导致骨髓组织经历典型的凋亡坏死、空虚、再生修复和恢复4个阶段的病理改变,但复合组的病变轻于电离组,恢复也更快。脾脏系数结果显示,照后9d、12d复合组脾脏系数显著高于电离组(p<0.05)。脾脏病理显示,脾损伤病变过程与骨髓造血组织基本相似,即早期以淋巴细胞凋亡为主,伴随脾小体不同程度的萎缩,中晚期淋巴细胞开始增生、间质纤维组织增生修复;复合组病变轻于电离组。神经细胞体外实验结果(1)4mW/cm2和6mW/cm2微波辐射组细胞增殖活性显著下降。6mW/cm2微波辐射增强电离辐射引起的细胞增殖活性下降的程度。单独微波组细胞的克隆形成率下降。4mW/cm2和6mW/cm2微波辐射增强电离辐射引起的细胞克隆形成率下降的程度。(2)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。4mW/cm2和6mW/cm2微波辐射增强电离辐射诱导的细胞凋亡率。微波与γ射线对细胞凋亡率存在协同促进作用。(3)各单独微波组SOD活性有上升趋势,但各组间没有统计学差异。单独微波组中,6mW/cm2微波辐射组的MDA含量上升。微波辐射能增强电离辐射引起的MDA含量上升,6mW/cm2微波辐射增强电离辐射引起的SOD活性下降。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。电磁辐射对神经行为影响的动物实验结果:(1)与对照组相比,1.5 mW/cm2组、6 mW/cm2组、12 mW/cm2组小鼠活动的总路程和活动时间增加,休息时间缩短,潜伏期明显缩短,错误次数增加(p<0.05)。但是3 mW/cm2组小鼠各指标没有显著变化。(2)各照射剂量微波辐射均未引起小鼠海马组织出现病理性改变。(3)与对照组相比,1.5 mW/cm2组、6 mW/cm2组、12 mW/cm2组小鼠海马组织中Hsp70 mRNA表达增加(p<0.05),但是S3组小鼠海马组织Hsp70 mRNA表达没有显著差异。人群调查结果:(1)基站周围电磁辐射强度未超过国家标准规定的一级标准。(2)调查结果显示,观察组和对照组人群在神经衰弱症状方面无显著性差异,未观察到移动基站微波辐射引起神经衰弱症候群发病率的增高。但随着暴露时间(居住年限)的延长,头痛、头晕、记忆力减退这三项指标的异常率有所增高(p<0.05),说明移动基站微波辐射对人体的健康危害与暴露时间仍有一定的关联。(3)观察组情感状态测试中紧张-焦虑、忧郁-沮丧、愤怒-敌意、疲惫-惰性和半结构投射实验中思、怒、惊得分显著高于对照组(p<0.05),心算、记忆扫描、连续识别、数字检索、曲线吻合等项目得分显著低于对照组(p<0.05),其中记忆扫描、连续识别、数字检索、目标追踪与居住年限呈负相关(p<0.05)。结论:1.低剂量的微波辐射可在一定程度上减轻γ射线对小鼠造血系统的急性损伤。低剂量微波预处理可刺激造血因子表达,促进骨髓造血细胞(尤其是粒系细胞)增殖,减轻γ射线对造血细胞的增殖抑制作用,同时降低损伤细胞的凋亡率,从而促进造血重建。2.在细胞水平上,微波和γ射线对人神经胶质瘤细胞的增殖抑制和氧化损伤有协同作用。联合照射对大鼠原代神经胶质细胞的增殖和凋亡无明显影响,仅可使ATP酶活性增高。在动物整体水平上,微波辐射可引起小鼠学习记忆能力下降和神经行为改变,并导致海马组织热休克反应。3.人群调查发现,基站微波辐射可影响附近居民的情感状况、智力、记忆学习、感知和心理运动神经行为的能力,导致负性情绪增加;随着时间的延长,神经衰弱相关症状的异常率有所增高。提示微波辐射对居民健康的影响不容忽视。更多还原

【Abstract】 Objective To study the joint biological effects of different doses of microwave radiation andγ-ray on the nervous system and hematopoietic system, as well as the protective effects of low doses of microwave radiation on theγ-ray-induced damage of hematopoietic system at the cellular and organism levels, to provide with imformation in exploring molecule mechanisms of combined effects of microwave andγ-rays.Methods Primary cultured bone marrow cells were exposed with microwave radiation,γ-ray radiation, as well as combined radiation of microwave andγ-ray, changes of cell proliferation and cell cycle were analyzed. Kunming mice were treated with a separate microwave radiation, a separateγ-ray radiation, as well as combined radiation of microwave andγ-ray. According to changes of animal survival rate, survival time, as well as peripheral blood cell count, animal model was set up for microwave radiation scheme which could effectively reduce the hazards of ionizing radiation; bone marrow nucleated cells(BMNCs) were counted, number and proliferative ability of hematopoietic stem/ progenitor cells were detected, and histopathological changes in the bone marrow were observed,GM-CSF and IL-3 levels in blood serum were analyzed using ELISA assay method.SHG44 human glioma cells were first randomly divided into 4 groups including Group control (C), Group ionizing radiation ( I), Group microwave(M) and Combined group (M + I). Group M + I samples received an initial exposure to microwave radiation and then subsequently to ionizing radiation. Standard MTT assay was used to observe the proliferation activity of the SHG44 human glioma cells. Changes in cell cycle and apoptosis were measured by means of flow cytometry. The activity of the superoxide dismutase (SOD) and malondialdehyde levels (MDA) were determined by applying commercial SOD Test Kits and MDA Test Kits, with procedures according to instructions of the manufacturer. Total RNA was extracted from each sample, performed with a commercial kit according to the manufacturer’s protocol. The level of Hsp70 expression was analyzed by a RT-PCR procedure. HSP70 protein level was analyzed using western blot analysis. Primary cultured rat glial cells were exposed with microwave radiation,γ-ray radiation, as well as combined radiation of microwave andγ-ray, cell proliferation and apoptosis were analyzed. Kunming mice were irradiated with 1.5mW/cm2, 3mW/cm2, 6mW/cm2 and 9mW/cm2 900MHz microwave, Changes of learning and memory ability after radiation were measured by open field test and step-through test. The levels of Hsp70 were detected with RT-PCR.Two hundred and four residents near and around a base station have been investigated. The intensity of electromagnetic field near the base station was measured. General information correlated with the investigation was collected by questionnaire. Neurobehavioral functions of the residents were examined by the B.NESC assessment system.ResultsPre-exposure of 120μW/cm2 900MHz microwave significantly improved the survival rate ofγ-ray irradiated mice, promoted the recovery ofγ-ray induced decline of peripheral white blood cells, improved spleen and thymus idex, enhanced BMNCs proliferative activity, promoted CFU-GM colony formation, and stimulated the hematopoietic growth factor GM-CSF, IL-3 expression ofγ-ray irradiated mouses. Bothγ-ray radiation alone and combined radiation led to pathological changes of bone marrow, which experienced four stages : apoptotic necrosis, emptiness, regeneration and repair, and restoration, but pathological damages of mice in combined group were less severe, and recovered faster than that of ionizing group. Spleen index of mice in combined group was significantly higher than that ofγ-ray group 9d, 12d after irradiation (p <0.05). Splenic pathological change was similar with that of bone marrow. Splenic pathological injuries in combined group were lighter than that ofγ-ray group.4mW/cm2 and 6mW/cm2 microwave radiation decreased proliferation activity and cloning efficiency of SHG44 human glioma cells.Combination of Microwave andγ-ray showed synergistic effect on both cell proliferation activity and the cloning efficiency.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 on apoptosis rate. The MDA level increased in all combination groups,and the SOD activity decreased in the group with combined radiation ofγ-ray and 6mW/cm2 microwave.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 for SHG44 human glioma cells.Proliferation of primary cultured rat glial cell inγ-ray group and combined radiation group withγ-ray and 6mW/cm2 microwave decreased slightly, and the ATPase activity was significantly increased.In animal experiment for neurobehavial effect, the time and total distance of movement at 1.5 mW/cm2, 6 mW/cm2 and 12 mW/cm2 microwave group increased, with a decrease in rest and latency time and a high frequency of mistake(p<0.05). The levels of Hsp70 in hippocampus of 1.5 mW/cm2, 6 mW/cm2 and 12 mW/cm2 microwave group were significantly increased (p<0.05).The intensity of electromagnetic radiation from this base station was below the national standard. No significant difference was found between the exposure group and the control group in the neurasthenia symptom. However, as the time of exposure extended, abnormal incidences of headache, dizziness, and memory drop increased (p<0.05). In addition, exposure of electromagnetic radiation could lead to an increase in scores of profile of mood state (POMS) test and semi-structural project test.Conclusions1. Low dose of microwave radiation could reduceγ-ray induced mice hematopoietic damages to some extent.2. 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.900MHz microwave radiation could impair neurobehavioral of mice such as learning and memory ability. The over expression of Hsp70 could be an early index of neuron functional damage induced by microwave.3. Microwave radiation from the mobile base station could affect the emotional status of the residents nearby.更多还原