【作者】 庞轶兵；

【导师】 郭国祯；

【作者基本信息】 中国人民解放军第四军医大学 ， 放射医学， 2003， 硕士

【摘要】 微波是一种频率范围在300MHz～300GHz之间的非电离辐射，广泛应用于雷达、航空、通讯、工业及医学等领域。近些年，随着频率为2450MHz家用微波炉的应用，及频率范围为800～1800MHz移动电话的普及，人们暴露于微波辐射的机会明显增加，公众已经开始关注暴露于微波辐射可能产生的生物效应及其对健康的影响。 微波的生物学效应主要有两种：热效应和非热效应。文献报道较多的是关于比吸收率(specific absorption rate，SAR)较高时微波辐射产生的热效应，而对非热效应报道相对较少。热和热效应的产生和微波的频率、功率密度、脉冲调制有关。一些流行病学及动物实验研究提示，微波辐射可能具有致癌、协同致癌或促进肿瘤发生的作用。有关2450MHz微波辐射的潜在基因毒性，尽管报道较多的是阴性结果，但确实也有一些阳性发现，例如：双着丝点染色体、无着丝点片段、微核数目的增加及DNA片段重排和基因表达改变等。 第四军医大学硕士学位论文 本实验旨在通过研究不同辐照强度和辐照时问的微波作川后，小鼠外周血．和脑组织中分于和蛋白水平的变化，来探讨微波所致小鼠脑组织细胞凋亡及其作用机制，以期为微波辐照的合理应用与防护提供一定的理论依％。 研究内容与结果： 1．凋亡过程中的细胞，其染色质DNA的断裂多数情况下是单链断裂，当DNA两条链上的切口相距不足12hp时就会发生双链断裂。根扼此原理采用 DNA聚合酶一 IKlenow大片段介导的生物素标记的dATP缺日木端标记方法（KLENOW法）和末端脱氧核昔酸转移酶介导的dUTP缺口末端标记方法（TWEL法）分别检测小鼠脑组织DNA单链和双链的损伤情况，（从而可以特异性的检测出小鼠脑组织中的凋亡细胞）。结果显示小鼠脑组织中 KLENOW和 TU’NEL阳性细胞数明显增加。 2．在诸种细胞凋亡的诱因中，体内代谢或外源性因素产生的活性氧自山基被证实可诱导细胞凋亡。超氧化物歧化酶（SOD）是清除氧自由基最重要的酶之一，它的活性反映了机体清除氧自山基的能力。谷脱甘肽 （GSH）是细胞内主要的非蛋白琉基，具有多种重要生理功能，事实上是一种氧化还原反应缓冲剂，能够通过亲核反应预防氧化损伤，清除射线所产生的活性氧自山基。活性氧自由基可引起脂质过氧化反应损伤细胞膜，并进而导致细胞死亡，其终产物丙二醛（MDA）的含量办间接反映了机体细胞被自由基损伤的程度。本实验对脑组织中SOD活性采用NBT羟胆法，GSH含量采用DTNB显色法，MDA含量采用TBA比色法测定。结果微波辐照后 SOD活性和 GSH含量显著降低，MDA含量显著增高，并且在小脑和大脑组织中自山基的变化水平和趋势不是完全一致。 3，细胞凋亡具有复杂的分子调控机制，许多基因都参与凋亡的发生。c－fos心Ciun作为凋亡促进基因与DNA结合具有直接调节转录活性的作用， －3－ 第四军医大学硕士学位论文其本身的活性和表达受到自山基、Cay离于、蛋白激酶等许多分子的调控。本实验采用V七stern印迹方法和免疫组织化学方法检测小鼠海马和小脑组织c－fos、叫un即早基回表达水平。发现微波辐照后其蛋白印迹明显增粗，免疫阳性细胞明显增多。 4回 次黄嘿吟鸟嘿吟磷酸核移酶（＊＊RT）基回是一种辐射敏感基囚，当受到一定强度的电离辐射后会发生基因位点突变，且这种突变是不可逆的，并在体内长期存在。本实验采用多核细胞法检测小鼠外周血淋巴细胞受到非电离辐射后其HPRT基因突变情况。随着辐照时间的延长和辐照强度的增大，HPRT基因的突变频率呈上升趋势，30 mw儿m‘辐照 7d组与对照组相比突变频率有显著差异。结论： 1．微波辐照可致小鼠脑组织细胞凋亡。 2．微波辐照可导致小鼠脑组织内自山基的产生与清除失衡；C－FOS和C－J：fl蛋白表达增加：DNA链断裂增加。 3．自由基在小脑和大脑组织中的变化水平和趋势不是完全一致，其原因可能与不同组织对辐射的敏感性不同有关。 4．微波辐照可致小鼠外周血淋巳细胞HPRT基因的突变频率增加，且与辐照强度和辐照时间呈正相关。 5．＊PRT基因可能是一种凋亡相关基因。更多还原

【Abstract】 Microwave in the frequency range 300MHz ~ SOOGHz, has long been used in radar, space research, telecommunications, industrial processing and medicine. In recent years, a large increase in the number of people who are potentially exposed to microwave radiation occurred with the introduction of household microwave ovens (2450 MHz) and mobile communications devices (800 ~ 1800 MHz). Public attention has been drawn to the possible biological effects and health hazards from exposure to microwave radiation.The biological effects of microwave are divided into two types: thermal and non-thermal effects. Thermal effects are associated with heat generated during microwave radiation when the specific absorption rate (SAR) is high. Lower intensity exposures that do not invoke thermoregulation are referred to as non-thermal which was less reported. Thermal and thermal induced-5-bio-effects correlated with some parameters of microwave such as frequencies, power densities, and pulsed modulations. Some epidemiological studies and in vivo experiments suggested an association between the incidence of cancer and exposure to microwave radiation. About the genotoxic effects of 2450 MHz microwave radiation, the majority data reported were negative. For the positive data, showed increasing the incidence of dicentric chromosomes, acentric fragments and micronuclei, DNA fragment rearrangement and gene expression alteration.In order to provide some evidences for health hazards from exposure to microwave radiation, in this study, HPRT gene mutation, free radicals level, protein expression and DNA breakage were investigated after the mice exposured to different intensity and time microwave radiation. RESEARCH CONTENTS AND RESULTS:1. When cells go through apoptosis, usually DNA single-strand breakage occurs. DNA double-strand breakage take place when the distance between two nicks of double strands is less than 12bp. According to this principle, we observed DNA single and double-strand breaks in brain tissue of mice, using the klenow fragment of DNA polymerase I -mediated nick end labeling method (KLENOW method), and terminal deoxynucleotidy I transferas?mediated dUTP nick end labeling (TUNEL method) . The result showed that the damage of DNA single and double-strand breaks in brain tissue of mice increased significantly.2. Reactive oxygen species (ROS) is one of the factors which induce apoptosis. ROS was generated during internal metabolism or stimulated by external factors. Superoxide dismutase (SOD) is one of the most important-6- enzymes that remove oxygen free radicals. The activity of SOD reflects the ability of the body removing oxygen free radicals. Glutathione (GSH, T-i -glutamyl-1 -cysteinylglycine) is an introcelluler non-protein thiol, which has various physiological functions, for example to preventing oxidative damage by nucleophilic reactions as a redox buffer, and removing reactive oxygen species produced under pathological conditions. ROS may damage cell membrane by lipid peroxidation. The essential metabolite MDA, reflects the level of cell damage. In this study, SOD activity, contents of GSH and MDA in brain tissue were determined by NBT method, DTNB method and TBA methods respectively. We found the activity of SOD and the content of GSH significant decreased, while the content of MDA in brain tissue significant increased after exposured to microwave radiation compared with that of control. The level of free radical was not completely the same in tissue of cerebellum and cerebrum.3. Apoptosis was mediated by many genes and proteins, were expressed in cells nucleus and promote the production of apoptosis. It was also reported that Fos/Jun could modulate DNA in transcription level. The activity and expression of Fos and Jun proteins were regulated by free radical, Ca2+ ion, protein kinase and so on. Western bloting and immunohistochemistry were used to assess the expression of the immediate early genes, c-fos and c-jun, in hippocampals and cerebellum of mice. Significant i更多还原