摘要
近年来有关移动电话微波辐射对人体健康的影响问题越来越引起人们的重视。国内外研究资料表明，移动电话微波辐射会对生物体产生广谱的生物效应，尤其是其非致热效应，但人们对移动电话微波辐射产生的非致热效应机理了解甚少。从分子水平来了解移动电话微波辐射的损伤机制和生物学行为，这不仅能维护广大移动电话使用者的健康，也为合理使用移动电话提供了实验依据。乳酸脱氢酶(lactatedehydrogenases，LDH)在中枢神经元中大量存在，其化学和生物学性质十分稳定。通常情况下神经细胞的LDH流出很少，而受损神经元流出的LDH则明显增大。细胞凋亡(apoptosis)，又称程序性细胞死亡(programmed cell death,PCD)，它是一个需要基因表达的主动过程，与凋亡关系最为密切的基因有Bcl-2(B cell lymphoma／leukemia2)和Bax(Bcl-2 associated X)。Bcl-2基因编码26KD的膜蛋白，主要存在于线粒体外膜上，Bcl-2的过量表达能抑制多种因素诱导的细胞凋亡。Bax基因编码一个21KD的蛋白，Bax蛋白以同源二聚体的形式存在，但Bax的作用与Bcl-2完全相反，Bax／Bax二聚体在体内过量表达时，可促进细胞凋亡。本课题采用神经细胞培养和整体动物相
浙江大学硕士学位沦文
结合的实验方祛，在对体外新生大鼠大脑皮层神经细胞进行原代培养的基础上，以频
率900MHz，功率密度分别为 0025mw／bm‘、0050mwtom’、0．100mwicm‘的模拟移
动电话微波辐射4、巴、12、16、20、24小时。检测LDH活性及细胞死亡率。整体动
物选用健康雄性SD大鼠，用频率为900MHz，功率额为0．050mwfom‘的模拟移动
电话微波每天上午和下千各辐射2小时，连续刀天。用免疫组织化学方法检测辐射
后相应时闷的脑组织中BclZ、Bax t白的表达情况。通过比较、分析LDH活性、细
胞死亡率以及SCI－2、B。蛋白的表达与移动电话微波辐射的关系，初步探讨长期移动
电话微波辐射对大鼠神经系统损伤作用的机理。
材料与方法
（一）细胞培养
1、皮层神经细胞培养：皮层神经细胞培养：新生大鼠断头取皮层，在37C、025％
胰酶消化液中剪碎，消化30分钟，离心后弃上清液、加入培养液中反复吹打，调细胞
密度为旷／d接种在经多聚赖氨酸前处理的培养板上。37℃、5％＊0。培养箱中培养，
3天后加入阿糖胞昔达10umol作用48小时以抑制非神经元增殖，以后每周换液2次c
培养至第12天，很据实验设计，进行下一步试验。
2、微波辐射参量：频率为900Mth，功率密度分别为 0．025mwfom‘、OD50mwtom‘、
0 100thwboZ
3、辐射方法：在细胞培养箱内，将微波辐射探头直接照射细胞培养板，每组按
辐射时间分组分别辐射4、8、12、16、20、24小时。
4、检测：用比色法测定培养液中LDH活性。神经细胞死亡率用苔盼蓝排斥试验
测定。
5、结果判定：在倒置相差显微镜下汁数500个细胞，着淡蓝色为死细胞，计算
2
汾江大学项士掌位论文
着色细胞的百分比即细胞死亡率。
（二）整体勾物实验
1、动物分组：80只健康雄性SD大鼠随机分为4组，分别为空白对照组、去颅
骨对照组、正常辐射组和去颅骨辐射组，每组20只。
2、去颅骨过程：大鼠经盐酸氯胺酮（100。g＆g）腹腔麻醉，用牙科磨钻在矢状
缝后 1．snun，中线旁 2 smlll处钻一直径 5。骨圆为避免手术的影响，待 72小时
后再行下一步实验。
3、微波辐射参量：频率为900MHz，功率密度为0050mwto‘。
4、微波辐射过程：正常辐射组和去颅骨辐射组每大上午和下午各辐射2小时，
连续ZI天。空白对照组、去颅骨对照组不接受微波辐射，余处理相同。
5、取衬：微波辐射结束后0小时、24小时、72小时和168小时（7无）断头处
死空白对照组、去颅骨对照组、正常辐射组和去颅骨辐射组大鼠各5只，将脑组织制
成石蜡标本，切片备用。
6、检测：采用SP法进行BclZ、BSX的免疫组织化学染色。
7、观崇记录：在m，mpus－CH光学显微镜下观察Bcl－2、Bax免疫反应的结果，
并计数，计算出平均阳性率（LI）。
（三）统计学处理
所有实验结果用均数土标准差＊上5）表示，各组之间的比较采用随机区组设计
资料的方差分析，所有数据在SPSS10．0软件包上处理。
结 果
一、和对照组相比，0．0501llw／Cm‘功率密度组辐射12小时、0．100mwlcm‘功率密
度组辐射8小时后均可引起神经细胞LDH活性和细胞死亡率增高（P＜0刀1），且随着辐
3
浙江大学硕士学位论又
射时间的延长，LDH活性和细胞死亡率匕逐渐上升，0．025mw／cm‘功率密度组未见明
显改变（P＞0．05）。
二、在空白对照组大鼠脑组织中，Bax亦有阳性表达，去颅骨辐射组大鼠中在辐
射结柬后早期阳性表达明显增加，去颅骨?
The increasing use of handportable mobiletelephone has raised concerns about possible health hazard effects.lt was well known to us, the handportable mobiletelephone microwave radiation caused broad-spectrum biological domino effect to organism ,especially its non-thermal effect.But the mechanisms of non-thermal effect has not been elucidated. From molecule level to realize the radiation traumatic mechanism and biological effect of handportable mobiletelephone microwave, it would not only maintenance^ the health of greatness handportable mobiletelephon consumer, but also provided experimental data about useing handportable mobiletelephone rightly. Measurement of lactate dehydrogenase (LDH) activity released to the extracellular bathing media has been found to be a simple yet quantitative method for assessing neuronal cell injury in cortical cell culture. Extracellular LDH was both chemically and biologically stable; the magnitude of LDH efflux in the cultures correlates in a linear fashion with the number of neurons
damaged by microwave exposure. Apoptosis was necessary in development of vital process and is very important in maintaining normal physiological function of organism. Bcl-2 (B cell lymphoma/leukemia 2) and Bax (Bcl-2 associated x) were the regulator genes in apoptosis and played a important role in the occurrence of apoptosis. Bcl-2 encoded a 26KD protein, which localized to outer mitochondrial membranes. Overexpression of Bcl-2 can blocked occurrence of apoptosis. Bax encoded a 21 KD protein. Bax protein could form homodimer. On the contrary, overexpression of Bax could promote occurrence of apoptosis. The task adopted cell culture combined with animal experiment. The neuron were radiated with microwave ,which was frequency of 900MHz and power density of 0.025 mW/cm2, 0.050mW/cn^ 0.100mW/cm2 for 4, 8> 12 ^ 16^ 2Q* 24hours,and the LDH in the media and the activities of neurons were assayed. The healthy male SD rats were radiated with microwave ,which was frequency of 900MHz and power density of 0.050mW/cm2 for 2 h each morning and afternoonjasting 21 days. Then immunohistochemisty were used to detect Bcl-2 ^ Bax expression in all the brain tissue.
Materials and methods
(? cell culture
l.Cell culture Primary dissociated cell cultures were prepared from cerebral cortex of fetal rats.The tissue was dissected, incubated in 0.25% trypsin for 30min.The isolated brain cells were plater at a density of 1 X 106/ml in L-polylysine-coated 12 -well plates.Cells were maintained in growth medium
consisting of 85% DMEM,15% fetal bovine serum at 37癈 in 5%CO2/humidified atmosphere.The medium was changed twice weekly^
2.Microwave paramete: frequency of 900MHz and power density of 0.025 mW/cm\ 0.050mW/cm\ 0.100mW/cm2.
3.Radiation method Experiments were performed in 10-12day- old cultures. In cell culture case, the neuron were radiated with microwave for 4 ^ 8> 12> 16^ 20? 24hours.
4.Detection: LDH level were measured by colorimetry test and the activities of neurons were measured by trjpan blue straining test(TBST).
5.Observation: Then we taken count of 500 cells in convert discrepancy microscope. The pigmentation cell was death.we accounted the percentage of pigmentation cell is the cell mortality (JH) Animal experiment
1. Animals: 80 healthy male SD rats (Body weight: about 200g) were divided into 4 groups in random: the group without treatment, the group of cranial defect, the group of normal radiation and the group of cranial defect radiation.
2.Craniectomies ; 2 groups were opened a bone window (diameter.4.5mm) in left parietal bone.To avoid the operation's influence,the next experiment would taken after 72 hours.
3, Microwave paramete. frequency of 900MHz and power density of 0.050mW/cm2.
4x Radiation method: the group of normal radiation and cranial defect radiation were radiated for 2 hour each morning and afternoon,lasting 21 days.
5x Material: 5 rats were killed respectively in four groups at CK 24 > 72^ 168hours (7 days) after radiation. Th
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