【作者】 许商成；

【导师】 余争平；

【作者基本信息】 第三军医大学 ， 劳动卫生学， 2010， 博士

【摘要】 线粒体功能障碍（mitochondrial dysfunction）,包括氧化磷酸化异常、能量代谢障碍、线粒体氧化应激及线粒体DNA（mitochondrial DNA, mtDNA）氧化损伤,是导致神经系统诸多疾病发生和发展的重要病因。电磁辐射和含镍材料的广泛运用在给生产生活带来便利的同时,所引发的环境污染和健康危害日益受到人们的关注。中枢神经系统是电磁辐射暴露和镍损伤的主要靶器官。基于线粒体功能障碍在诸多中枢神经系统疾病中的重要作用,本研究以线粒体为靶细胞器,探讨电磁辐射和镍暴露致神经细胞损伤的机制,并寻找相应的生物学防护措施。实验中,我们以原代培养的皮层神经元、人源性的神经母瘤SH-SY5Y细胞和小鼠神经母瘤Neuro2a细胞为研究模型,将其暴露于1800MHz射频段电磁辐射和不同浓度的氯化镍（NiCl₂）中,做了如下两部分的实验:一、电磁辐射对神经细胞线粒体的氧化损伤及Tfam的保护作用（一）电磁辐射辐照对皮层神经元线粒体的氧化损伤电磁辐射辐照后6h、12h、24h和48h,皮层神经元内活性氧ROS水平显著升高,细胞活性显著降低,ATP含量和线粒体膜电位（mitochondrial membrane potential,△Ψm）明显下降。而且线粒体内8-羟基脱氧鸟苷（8-hydroxy-2-deoxy-guanosine,8-OHdG）含量显著升高,而mtDNA拷贝数和mtDNA编码基因（ND1、COX I和ND6）mRNA水平显著下降。提示电磁辐射辐照对皮层神经元线粒体造成明显的氧化损伤。（二）电磁辐射辐照对皮层神经元Tfam表达、转运和结合mtDNA活性的影响资料显示,导致线粒体功能障碍的原因很多,而mtDNA突变是其中最为重要的因素。Tfam是维持mtDNA完整性和线粒体功能的核心因子。在多种病理生理过程中,氧化应激可以通过影响Tfam的表达或活性导致线粒体功能障碍和细胞的氧化损伤。所以本部分研究从Tfam作用通路的角度,探讨电磁辐射致神经细胞线粒体功能障碍的可能的生物学机制。结果表明,电磁辐射辐照后,皮层神经元内Tfam mRNA和蛋白的表达显著性升高。转运分析（import assay）实验显示皮层神经元线粒体转运体外表达的S³⁵-Tfam蛋白的效率明显降低。凝胶迁移实验（EMSA）也表明线粒体内Tfam与mtDNA轻链启动子结合显著降低。表明电磁辐射辐照抑制了Tfam的转运,对线粒体内Tfam与mtDNA结合有影响。提示电磁辐射可能是通过抑制Tfam转运来造成神经细胞线粒体损伤的。（三） Tfam过表达在电磁辐射致SH-SY5Y细胞线粒体损伤中的保护作用1.成功构建Tfam表达质粒,转染SH-SY5Y细胞。电磁辐射辐照后24h,Tfam过表达能显著降低细胞内ROS水平,减轻电磁辐射对SH-SY5Y细胞活性的损伤,提高ATP含量,有效缓解电磁辐射辐照引起的SH-SY5Y细胞线粒体内8-OHdG含量的升高,维持mtDNA拷贝数和mtDNA编码基因（ND1、COX I和ND6）mRNA水平。提示Tfam过表达能有效改善电磁辐射辐照所致的神经细胞线粒体氧化损伤。2.敲除Tfam蛋白中具有转录活性的C末端,保留其维持mtDNA核样结构和mtDNA拷贝数的功能。电磁辐射辐照后24h,C末端缺失的Tfam（△C-Tfam）同野生型Tfam一样,能显著降低细胞内ROS水平,减轻电磁辐射对SH-SY5Y细胞细胞活性的损伤,提高ATP含量,有效缓解电磁辐射辐照引起的SH-SY5Y细胞mtDNA的氧化损伤。提示Tfam拮抗电磁辐射致线粒体氧化损伤的机制与Tfam维持mtDNA核样结构和mtDNA拷贝数的功能紧密有关。（四）β淀粉肽对神经细胞线粒体的氧化损伤及Tfam的保护作用β淀粉肽（Aβ）沉积是阿尔茨海默病发生和发展中的重要病理特征。体内外研究均证实,Aβ进入线粒体后,直接作用于线粒体蛋白,能明显引起线粒体功能障碍和氧化应激。为了进一步探讨线粒体功能障碍在电磁辐射致神经细胞损伤中的作用及Tfam的保护机制,我们选择用Aβ处理SH-SY5Y细胞造成线粒体功能障碍模型作为阳性参照。结果发现,电磁辐射辐照致神经细胞线粒体氧化损伤的效应与β淀粉肽十分相似,能明显引起神经细胞线粒体氧化损伤。野生型Tfam和缺失型ΔC-Tfam的过表达均能显著减轻β淀粉肽对SH-SY5Y细胞细胞活性的损伤,降低细胞内ROS水平,提高ATP含量,有效缓解β淀粉肽引起的SH-SY5Y细胞线粒体内8-OHdG含量的升高,维持mtDNA拷贝数和mtDNA编码基因（ND1、COX I和ND6）mRNA水平。二、镍暴露对神经细胞线粒体的氧化损伤及褪黑素的保护作用（一）镍暴露对神经细胞线粒体的氧化损伤将皮层神经元和小鼠神经母瘤Neuro2a细胞暴露于不同浓度的NiCl2 （0.125 mM, 0.25 mM, 0.5 mM和1 mM）12h后或0.5 mM NiCl2不同时间（0 h, 3 h, 6 h, 12 h和24 h）后,神经细胞内ROS含量显著升高,而且呈现剂量依赖效应关系。神经细胞线粒体功能明显受损,表现为线粒体脱氢酶活性,细胞ATP含量,线粒体膜电位和mtDNA含量显著降低。表明一定剂量的镍暴露造成了神经细胞线粒体的氧化损伤。（二）褪黑素预处理对镍暴露致神经细胞线粒体氧化损伤的保护作用用神经系统抗氧化剂和线粒体功能保护剂-褪黑素（1mM）预处理皮层神经元和Neuro2a细胞2h后,褪黑素能有效降低神经细胞的氧化应激,改善镍暴露引起的线粒体功能障碍和神经细胞活性下降。综上所述,电磁辐射辐照能够引起神经细胞氧化应激,造成线粒体功能障碍。并且电磁辐射致线粒体氧化损伤可能与电磁辐射抑制Tfam转运有关。而Tfam过表达能有效改善电磁辐射对mtDNA的氧化损伤,降低神经元的氧化应激,维持线粒体稳态。Tfam这种保护效应主要依赖于Tfam对mtDNA核样结构和mtDNA拷贝数的维护。与此类似,镍暴露能明显造成神经细胞线粒体的氧化损伤,而褪黑素预处理能显著拮抗镍的神经毒性。本研究表明,电磁辐射和镍暴露具有明显的神经毒性。线粒体功能障碍可能是电磁辐射和镍暴露所致神经细胞损伤的主要机制,而通过降低氧化应激和改善线粒体功能障碍能够有效缓解电磁辐射和镍暴露所致的神经毒性。通过研究,不仅能够揭示电磁辐射和镍暴露致中枢神经系统损伤的新机制,而且为电磁辐射和镍暴露致中枢神经系统损伤的防护提供有效线索。更多还原

【Abstract】 Mitochondrial dysfunction including oxidative phosphorylation efficiency、energy metabolism disturbance、mitochondrial oxidative stress and mitochondrial DNA （mtDNA） oxidative damage, is considered to make great contributions to various diseases in nervous system. The rapidly growing application of electromagnetic radiation （EMR） and nickel in modern technologies has raised considerable concerns about their potential hazardous effects on human health. It is reported that nervous system is one of the most important targets in the toxicity of EMR and nickel. The purpose of this study was to investigate whether the mitochondrial dysfunction was involved in the neurotoxicity of EMR radiation and nickel exposure. In addition, we would like to find some potential molecular or substances that could protect against the neurotoxicity induced by EMR radiation and nickel exposure. In order to address this issue, different kinds of nerve cells, including primary cultured cortical neurons, human neuroblasma SH-SY5Y cells and mouse neuroblasma Neruo2a cells, were exposed to 1800MHz radiofrequency radiation （RF） and various concentration of nickel. The main results and conclusions were as following:一、EMR radiation induced oxidative damage to mitochondria in nerve cells: the protective effects of Tfam（一） EMR radiation induced oxidative damage to mitochondria in cortical neuronsAt 6h,12h,24h and 48h after EMR radiation,ROS production obviously increased,the cell viability of cortical neurons significantly decreased,the content of ATP and mitochondrial membrane potential （△Ψm） markedly reduced. In addition, the contents of 8-hydroxy-2-deoxy-guanosine（8-OHdG） significantly elevated, while the mtDNA copy number and mtDNA transcripts （ND1、COX I and ND6） levels obviously decreased. It demonstrated that EMR radiation induced oxidative damage to mitochondria in cortical neurons.（二） EMR radiation effected the expression and import of Tfam, the ability of Tfam in binding mtDNA in cortical neuronsNumous studies indicate that mitochondrial dysfunction is largely attributable to mtDNA mutation. Tfam works as the key factor for the maintenance of mtDNA. In some pathophysiology situations, oxidative stress could cause the oxidative damage to mitochondria through disturbing the expression and activity of Tfam. Thus, in this part, we hypothesized that EMR radiation disturbed the expression and activity of Tfam,which might be related with the oxidative damage of mitochondria in radiated-nerve cells. As a result, we found that both the mRNA levels and protein levels of Tfam obviously elevated after EMR radiation. Additionally,import assay showed that EMR radiation decreased the import of S³⁵-Tfam from cytoplasm to mitochondria. Furthermore,electrophoretic mobility shift assay （EMSA） exhibited that the binding of Tfam to the mtDNA promoter significantly decreased after EMR radiation. It indicated that EMR radiation inhibited the import of Tfam and repressed the acticity of Tfam in binding mtDNA in mitochondria, which may lead to the oxidative damage to mitochondria in nerve cells.（三） Tfam overexpression protected mitochondria against oxidative damage induced by EMR radiation in SH-SY5Y cells1. We successfully constructed the plasmid expressing Tfam and transfected the recominated plasmid into SH-SY5Y cells. At 24h after EMR radiation,overexpression of Tfam could significantly reduce ROS production, reverse the decrease of cell viability, improve ATP content, effectively attenuate the oxidative damage of mtDNA induced by EMR radiaiton in SH-SY5Y cells. It suggested that Tfam overexpression protected mitochondria against oxidative damage induced by EMR radiation in SH-SY5Y cells.2. We disrupted the C-terminal tail of Tfam,which had no ability in promoting mtDNA transcripts but could still maintain the nucleoid structure of mtDNA and mtDNA copy number. As the same as the wild-type Tfam, disrupted Tfam also could significantly reduce ROS production, reverse the decrease of cell viability, improve ATP content, effectively attenuate the oxidative damage of mtDNA induced by EMR radiation in SH-SY5Y cells. It suggested that the protective effects of Tfam overexpression against EMR radiation-induced oxidative damage to mitochondria were largely attributed to the ability of Tfam in maintaining the nucleoid structure of mtDNA and mtDNA copy number.（四）β-amyloid induced oxidative damage to mitochondria in nerve cells: the protective effects of Tfamβ-amyloid deposition has been proposed as the major pathogenic event in the development and progression of Alzheimer’s disease （AD）. Oxidative stress and mitochondrial dysfunction have been the provital mechanism involved in Aβ-mediated neurotoxicity. Evidence suggests that Aβenters mitochondria and interacts with the mitochondrial proteins, induces mitochondrial dysfunction and causes oxidative stress. In order to further verify the involvment of mitochondrial dysfunction in the neruotoxicity of EMR radiation and the protective effects of Tfam, we established another oxidative damage model of mitochondria by administration of Aβ_1-42 as a positive control. As expected, Aβ_1-42 caused oxidative damage to mitochondria in SH-SY5Y cells, which was similar with that in irradiated-cells. Both Tfam with or without C-terminal tail could effectively reduce oxidative stress induced byβ-amyloid, attenuate the oxidative damage of mtDNA and reverse mitochondrial dysfunction.二、Nickel exposure induced oxidative damage to mitochondria in nerve cells: the protective effects of melatonin（一） Nickel exposure induced oxidative damage to mitochondria in nerve cellsAfter primary cultured cortical neurons and mouse neuroblastoma Neuro2a cell lines were exposed to different concentrations of nickel chloride （NiCl₂） （0.125 mM, 0.25 mM, 0.5 mM and 1 mM） for 12 h or 0.5 mM NiCl₂ for various periods （0 h, 3 h, 6 h, 12 h and 24 h）,we found that nickel significantly increased ROS production and caused the loss of cell viability both in cortical neurons and Neuro2a cells. In addition, nickel exposure obviously inhibited the mitochondrial function, disrupted the mitochondrial membrane potential, reduced ATP production and decreased mtDNA content. It indicated that nickel caused oxidative damage to mitochondria in nerve cells.（二） Melatonin pretreatment protected mitochondria against oxidative damage induced by nickel exposure in nerve cellsThe pretreatment of melatonin, a kind of efficient neuroprotective agents famous for its activity in reducing oxidative stress and maintaining mitochondrial function, efficiently attenuated the oxidative damages to mitochondria in nickel-treated nerve cells.In conclusion, our results demonstrated that EMR radiation induced oxidative stress and caused oxidative damage to mitochondria. It may be related to the inhibitory effects of EMR radiation on the import of Tfam in cortical neurons. In addition, Tfam overexpression effectively reduced oxidative stress and protected mitochondria against oxidative damage induced by EMR radiation in nerve cells. These protective effects were largely attributed to the ability of Tfam in maintaining the nucleoid structure of mtDNA and mtDNA copy number. Similarly, nickel exposure obviously induced oxidative damage to mitochondria, which were efficiently attenuated by melatonin pretreatment. All of our study indicated that EMR radiation and nickel exposure had deleterious effects on nerve cells through causing mitochondrial dysfunction. Importantly, it was potential to protect against the neurotoxicity of EMR radiation and nickel by reducing oxidative stress and maintaining mitochondrial function. It not only revealed the mechanism of neurotoxicity induced by EMR radiation and nickel exposure,but also provided the clues for protecting agaisnt EMR radiation and nickel exposure.更多还原