【作者】 刘仕昌；

【导师】 杨卓；

【作者基本信息】 南开大学 ， 生理学， 2012， 博士

【摘要】 纳米技术指的是将普通尺度的材料用特殊的工艺加工制作而成，使其具有纳米尺度并表现出许多不同的特性。由此加工而成粒径小于100nm的材料被人们称为纳米材料。由于纳米材料具有小尺寸效应、大的比表面积、光催化等特性，现在已经被广泛应用多个领域，例如医学、药物载体、农业以及工业生产等等。纳米二氧化钛（Nanosized titanium dioxide，nano-TiO2）作为最常用的纳米材料之一，在工业中被大量制作并应用于印刷业、造纸业、食物添加剂以及颜料、化妆品等产业。虽然纳米二氧化钛已被广泛应用，但是关于纳米二氧化钛对人体健康以及周围环境的影响，目前仍不十分清楚。纳米二氧化钛可以通过多种途径进入人体，例如通过呼吸道吸入、经由消化道摄入、通过皮肤直接渗入以及通过注射的方式进入到血液循环系统等等。这样纳米二氧化钛极有可能到达人体的各个器官，对人体健康产生影响。研究发现，由于具有纳米尺度及特殊的理化性质，纳米材料可以穿透血脑屏障进入大脑，因此关于纳米二氧化钛对中枢神经系统的影响的研究至关重要。本研究从整体水平、细胞水平以及离子通道水平研究了纳米二氧化钛对中枢神经系统的影响，并对其作用的可能机制进行了初步探讨。第一部分纳米二氧化钛对Wistar大鼠空间认知能力的影响目的：观察纳米二氧化钛对Wistar大鼠空间认知能力的影响，并对其可能机制进行探讨。材料与方法：大鼠随机分成三组：正常对照组、5mg/kg纳米二氧化钛组以及50mg/kg纳米二氧化钛组。采用腹腔注射的方式给予大鼠纳米二氧化钛悬浊液，每天一次，连续给予21天。之后，利用Morris水迷宫实验检测了纳米二氧化钛对大鼠空间认知功能的影响，利用电生理实验记录大鼠海马Schaffer侧枝至CA1区的长时程增强效应（long-term potentiation，LTP）。利用电感耦合等离子体质谱法（inductively coupled plasma-mass spectrometry，ICP-MS）检测腹腔注射纳米二氧化钛后大鼠海马区Ti元素的含量变化，同时用ELISA方法检测纳米二氧化钛对海马脑区超氧化物歧化酶（Superoxidedismutase，SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）活性的影响及丙二醛（malondialdehyde，MDA）含量的改变。结果：1、ICP-MS检测发现，给予大鼠腹腔注射纳米二氧化钛后，随着纳米二化钛浓度的增高，大鼠海马区的Ti元素含量逐渐增高，与正常组相比具有统计学差异。2、Morris水迷宫实验：在空间探索阶段，与正常对照组及5mg/kg纳米二氧化钛组相比，50mg/kg纳米二氧化钛组大鼠的逃避潜伏期明显增加，在定位巡航阶段，50mg/kg纳米二氧化钛组大鼠在目标象限游泳时间及穿越平台的次数显著减少，正常组与5mg/kg纳米二氧化钛组大鼠没有统计学差异。3、LTP实验：与正常对照组及5mg/kg纳米二氧化钛组相比，50mg/kg纳米二氧化钛组大鼠兴奋性突触后电位的斜率显著降低（P<0.05），而前两者没有统计学差异。4、 ELISA实验：50mg/kg纳米二氧化钛组及5mg/kg纳米二氧化钛组大鼠海马区的SOD、GSH-Px活性均显著下降，而MDA含量均显著增高，与正常组相比，具有统计学差异。结论：通过本部分实验可知，纳米二氧化钛可以通过血脑屏障，并在大鼠海马脑区沉积。同时纳米二氧化钛可以降低大鼠海马Schaffer侧枝至CA1区的LTP水平，引起大鼠空间认知功能受到损害，而这可能与纳米二氧化钛引起神经元发生氧化应激损伤有关。第二部分纳米二氧化钛对PC12细胞的损伤作用目的：利用PC12细胞作为神经元模型，观察纳米二氧化钛对神经元细胞的损伤作用。方法：将不同浓度的纳米二氧化钛与PC12细胞分别共培养6,12,24和48小时后，利用倒置相差显微镜观察PC12细胞形态的变化，用四唑盐比色法（3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide，MTT）检测纳米二氧化钛对PC12细胞存活率的影响。利用流式细胞术检测PC12细胞发生凋亡的情况和活性氧簇（reactiveoxygen species，ROS）在细胞内的积聚水平。同时，通过加入ROS清除剂2-巯基丙酰甘氨酸（N-(2-mercaptopropionyl)-glycine，N-MPG），观察ROS的产生对细胞凋亡的影响。结果：1、纳米二氧化钛与PC12细胞共培养后，细胞的形态学发生明显变化，细胞的数量也逐渐变少，具有浓度依赖性，同时，可以看到纳米二氧化钛在细胞表面聚集。2、MTT实验：与纳米二氧化钛共培养后，细胞的存活率显著下降，且具有时间和浓度依赖性。3、流式细胞术：检测发现纳米二氧化钛可以引起细胞内ROS水平的增高，且可以诱导PC12细胞发生凋亡。4、ROS清除剂N-MPG可以降低纳米二氧化钛对PC12细胞损伤，使PC12细胞的凋亡率降低。结论：纳米二氧化钛可能使ROS在细胞内积聚进而诱导PC12细胞发生凋亡现象。第三部分纳米二氧化钛对大鼠海马CA1区神经元离子通道的影响目的：利用膜片钳技术，观察纳米二氧化钛对大鼠海马CA1区神经元电压门控钠通道、钾通道及神经元兴奋性的影响。方法：利用全细胞膜片钳技术，在电压钳记录模式下，观察纳米二氧化钛对电压门控钠通道、钾通道的电流幅值及通道动力学的影响。在电流钳记录模式下，观察纳米二氧化钛对神经元动作电位的影响。结果：1、纳米二氧化钛抑制了电压门控钠通道电流的幅值，具有浓度依赖性，使稳态失活曲线右移，失活后恢复时间延长，但对其激活曲线没有显著影响。2、纳米二氧化钛抑制了瞬时外向钾电流及延迟整流钾电流的幅值，同时加快了瞬时外向钾通道的失活过程并使失活后恢复时间延长，但是对瞬时外向钾通道的及延迟整流钾电流的激活动力学没有显著影响。3、纳米二氧化钛降低了单个动作电位的峰值及超射值，增加了半峰宽及动作电位的发放频率。结论：纳米二氧化钛可以抑制电压门控钠通道和钾通道，并使神经元兴奋性增加，进而使神经元发生损伤。更多还原

【Abstract】 Nanotechnology involves the creation and manipulation of materialsat the nanoscale level to create unique products that exploit novelproperties. Nanomaterials range in size from1to100nm. The newphysical and chemical properties of novel engineered nanoparticle makethem extremely attractive for using in applications such as medicalscience, drug applications, agricultural and defense industries. Nanosizedtitanium dioxide （nano-TiO₂）, one of the most widely used nanoparticles,has now been produced in a large industrial scale and used widely inpaints, paper and plastics, as well as in food additives and colorants.Despite wide ranges of applications, there is a serious lack ofinformation on the impact of nano-TiO₂on human health and theenvironment. The extensive uses in medical research and industrialapplications highlight that there are many routes for the nano-TiO₂to potentially enter into human bodies, such as through inhalation（respiratory tract）, ingestion （gastrointestinal tract）, dermal penetration（skin） and injection （blood circulation）. With the ultrafine size andunusual properties, nano-TiO₂can enter human body and cross biologicalbarriers such as blood brain barrier （BBB） relatively unimpeded. Thereby,the studies to evaluate the effects of nano-TiO₂on the central nervoussystem （CNS） are allimportant.In this study, we investigated the effects of nano-TiO₂on the spatialcognition capability of Wistar rats by intraperitoneal injection.Meanwhile, the cytotoxicology of nano-TiO₂and the effects of nano-TiO₂on the ion channels of hipocampal CA1neurons were detected inexperiments. Part1the effects of nano-TiO₂on the spatial cognition capability ofWistar ratsObjective: To investigate the effects of nano-TiO₂on synaptic plasticityand spatial cognition of Wistar rats, and discuss the underlingmechanism.Material and methods: Rats were randomly divided into three groups:normal control group,5mg/kg nano-TiO₂group and50mg/kg nano-TiO₂group. Rats were treated intraperitoneally with different concentrations ofnano-TiO₂suspension once a day for3weeks. After that, the Morriswater maze （MWM） test was employed to evaluate the spatial cognitivefunction. The long term potentiation （LTP） from Schaffer collaterals toCA1region in the hippocampus was recorded. The Ti content in thehippocampus was tested by the inductively coupled plasma-massspectrometry （ICP-MS）. In addition, the activity of superoxide dismutase（SOD）， glutathione peroxidase（GSH-Px） and the content of malondialdehyde （MDA） in hippocampus were detected to approach thepossible mechanism of neuron damage caused by nano-TiO₂.Results:1. The ICP-MS test showed that the Ti content in the hippocampus wasincreased in a concentration-dependent manner when rats wereexposed to nano-TiO₂, which indicated that nano-TiO₂could crossBBB and accumulate in the hippocampus region.2. The MWM test showed that the escape latencies in place navigationphase was significantly increased in50mg/kg nano-TiO₂groupcompared to those of5mg/kg nano-TiO₂group and the control group（P<0.01）. Moreover, in spatial probe phase, the time percentage intarget quadrant was significantly decreased in50mg/kg nano-TiO₂group （P<0.05）; there was no significance between5mg/kgnano-TiO₂group and control group. And the number of platformlocation crossings was also decreased in both the50mg/kg nano-TiO₂ group and the5mg/kg nano-TiO₂group compared to that of controlgroup.3. The LTP test indicated that the slope of excitatory postsynapticpotential （EPSP） was lower in50mg/kg nano-TiO₂group than that of5mg/kg nano-TiO₂group and control group （P<0.05）.4. In50mg/kg nano-TiO₂group and5mg/kg nano-TiO₂group, theactivity levels of superoxide dismutase （SOD） and glutathioneperoxidase （GSH-Px） were significantly decreased while the contentof malondialdehyde （MDA） was significantly increased comparedwith those of control group.Conclusion:Nano-TiO₂can cross the BBB and accumulate in the hippocampus.Meanwhile, nano-TiO₂could decrease LTP from Schaffer collaterals toCA1region in the hippocampus and impair the spatial cognitivecapability of rats, which may partly due to oxidative stress damage of neurons caused by nano-TiO₂. Part2cytotoxicolgy of nano-TiO₂on PC12cellsObjective: To investigate the cytotoxicology of nano-TiO₂on PC12cells.Methods: PC12cells were cultured with different concentrations ofnano-TiO₂for6,12,24and48h. The inverted phase contrast microscopewas used to observe the morphology of nano-TiO₂treated PC12cells,and the cellular viability was tested by3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Furthermore, the flowcytometry was used to detect the apoptosis of PC12cells and the level ofintracellular reactive oxygen species （ROS） after cells were treated bynano-TiO₂. The ROS scavenger N-（2-mercaptopropionyl）-glycine （N-MPG） was used to approach the relationship between the cellapoptosis and intracellular ROS accumulation.Results:1. When PC12cells were incubated with different concentrations ofnano-TiO₂for24h, there was a significant change in the cellmorphology, and the cell number was reduced in aconcentration-dependent manner.2. Results of MTT assay showed that the cellular viability wassignificantly decreased in concentration and time dependent mannerafter cells were treated with nano-TiO₂.3. Results of flow cytometry test: The treatment of nano-TiO₂induced theapoptosis of PC12cells and increased the level of intracellular ROS.4. The ROS scavenger N-MPG improved the cellular viability andreduced the damage caused by nano-TiO₂. Conclusion:Nano-TiO₂could cause the accumulation of intracellular ROS andinduce the cell apoptosis successively. Part3the effect of nano-TiO₂on voltage-gated ion channels in rathippocampal CA1neuronsObjective: To approach the effect of nano-TiO₂on voltage-gated ionchannels in rat hippocampal CA1neuronsMethods: The effects of nano-TiO₂on voltage-gated sodium currents （INa）and voltage-gated potassium currents were studied in rat hippocampalCA1neurons using the voltage clamp technique in the whole-cellconfiguration. Meanwhile, the effects of nano-TiO₂on the actionpotential （AP） were detected by the current clamp technique in thewhole-cell configuration.Results:1. The amplitude of INawas significantly inhibited by nano-TiO₂in aconcentration-dependent manner, which produced a positive shift inthe inactivation curve of INaand delayed the recovery of INafrominactivation. The activation curve was not affected. 2. The amplitude of transient outward potassium current （IA） and delayedrectifier potassium current （IK） was significantly inhibited bynano-TiO₂. Meanwhile, the steady-state inactivation curve of IAwasshifted to the left by nano-TiO₂, and the recovery of IAfrominactivation was delayed. There were no significant shift in theactivation curve of IAand IKafter nano-TiO₂treatment.3. In the present of nano-TiO₂, the peak amplitude and overshoot of theevoked single action potential were decreased, but the half-width wasincreased. Furthermore, the firing rate of repetitive firing wasdecreased.Conclusion:Nano-TiO₂could alter the excitability of neurons by impairingfunctions of voltage-gated sodium channels （VGSCs） and Kv, andthereby induce damages of neurons.更多还原