【作者】 章军；

【导师】 姚耿东； 朱心强；

【作者基本信息】 浙江大学 ， 劳动卫生与环境卫生学， 2007， 博士

【摘要】 背景和目的目前纳米技术正在世界范围内迅速发展，各种新型纳米材料正逐渐被广泛应用于多个领域，纳米材料的研究者、生产者、消费者和纳米废物处理者接触纳米材料以及纳米材料进入生态环境的机会也越来越多，其生物安全性还不确定，所以纳米材料的生物安全性研究受到了各国学者、有关管理部门及社会公众的广泛关注。纳米材料可以穿透血脑屏障、血眼屏障及血睾屏障，从而可能危害到生物体的安全。一些纳米材料如单壁纳米碳管（SWCNT）和Fe₂O₃-Glu纳米颗粒可以达到动物的多种器官组织，如眼、心、肝、脾、肺、肾、胃、脑、骨骼、肌肉、小肠、皮肤、血液及性腺组织。研究表明纳米材料可以在细胞水平、亚细胞水平、基因、蛋白水平及整体动物水平对生物体产生影响。与同种物质微米级的颗粒比较，一般纳米颗粒的生物活性更强，对机体的潜在危害也可能更大。流行病学研究表明，室外空气污染物中可吸入颗粒物（PM）与心血管疾病的发病率和死亡率有关，而心血管疾病是严重威胁人类的健康与生命的重大疾病。因此，纳米颗粒对心血管系统的毒性问题格外引人关注。迄今为止，我们尚未发现纳米SiO₂对心血管系统影响的研究报道，目前的研究集中在肺部毒性方面，结果显示纳米SiO₂较微米二氧化硅可能有更强的脂质过氧化、损害抗氧化系统倾向，引起更严重的肺部急性损伤。鉴于纳米材料可能在心血管损害中起到非常重要的作用，本项目拟以纳米SiO₂为研究对象，采用体外、体内和人群研究相结合的方法，较全面的观察纳米SiO₂对心血管系统的影响。这样不仅可以从整体动物水平观察到纳米SiO₂对心血管主要功能的影响，而且可以从体外实验深入了解纳米SiO₂引起血管内皮细胞损伤的分子机制。方法和结果本课题分以下三部分进行研究：研究一：纳米SiO₂对人脐静脉血管内皮细胞（HUVECs）的毒性影响方法：设置生理盐水（NS）对照组、微米SiO₂对照组（剂量同纳米SiO₂高剂量）、纳米SiO₂低剂量组、纳米SiO₂中剂量组和纳米SiO₂高剂量组5个剂量组，DNA损伤检测增加10μg／ml MNNG阳性对照组。观察指标包括IC₅₀、细胞凋亡、细胞周期、DNA损伤以及炎症因子测定，处理时间均为24小时。具体如下：不同浓度纳米SiO₂（0～800μg／ml）处理HUVECs，采用台盼蓝排斥实验测定IC₅₀，以50％IC₅₀为纳米SiO₂高剂量；用Annexin V、PI双染色细胞凋亡试剂盒和细胞周期试剂盒在流式细胞仪上观察纳米SiO₂对HUVECs细胞凋亡和细胞周期的影响；DNA损伤检测：各组细胞经处理后用流式细胞仪和免疫荧光技术检测HUVECs细胞核γH2AX的焦点形成情况；炎症因子测定：HUVECs经纳米SiO₂处理后，用免疫试剂盒测定培养液中的肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）和高敏C反应蛋白（hsCRP）的释放情况。结果：用概率单位法计算纳米SiO₂作用于HUVECs 24小时的IC₅₀为194.9μg／ml。本试验中我们采用100μg／ml(约50％IC₅₀)作为纳米SiO₂的最高浓度。流式细胞仪检测显示，25μg／ml、50μg／ml和100μg／ml纳米SiO₂组作用HUVECs24小时后细胞凋亡率分别为10.5％、17.9％和15.3％，与100μg／ml微米SiO₂组（7.5％）和NS对照组（5.5％）相比均有显著性差异（p＜0.05）。100μg／ml微米SiO₂和各剂量组纳米SiO₂均显著引起了细胞G1期阻滞，S期也显著减少（p＜0.05），而各剂量组纳米SiO₂和微米SiO₂相比各期没有显著性差异（p＞0.05）；各剂量组纳米SiO₂均可引起HUVECs显著的DNA损伤，25μg／ml、50μg／ml和100μg／ml作用24小时后的γH2AX阳性细胞的百分率分别为14.2％、23.9％和25.9％，与100μg／ml微米SiO₂组（11.9％）和NS对照组（1.4％）相比均有显著性差异（p＜0.05）；各剂量组纳米SiO₂均可显著促进炎症因子TNF-α的释放，除25μg／ml组外，50μg／ml和100μg／ml纳米SiO₂组可以显著促进IL-6和hsCRP的释放。以上结果说明处理24小时后纳米SiO₂对血管内皮细胞存在损害效应。除细胞周期外，纳米SiO₂所引起的效应均比微米级SiO₂严重，说明SiO₂粒径尺度的改变对其毒性有显著影响。研究二：纳米SiO₂对大鼠心血管系统的毒性影响方法：根据预实验结果，设置NS对照组、微米SiO₂对照组（10mg／kg）、纳米SiO₂低剂量组（2.5mg／kg）、纳米SiO₂中剂量组（5mg／kg）和纳米SiO₂高剂量组（10mg／kg）5个剂量组，观察指标包括血压、心率、血栓形成时间、血小板计数和血小板聚集率。具体如下：1)对大鼠血压和心率的影响：动物麻醉后分离左颈总动脉（约2cm），血管插管接压力换能器，用Medlab-E生物信号采集系统测量受试材料对血压心率的影响。2)对血栓形成的影响：首先在Kura及唐映红方法的基础上构建了FeCl₃血栓模型，分离左侧颈动脉按照血压心率监测方法做颈动脉血管插管，MedLab-E生物信号采集处理系统监测血压和心率信号，各组处理时间均为4h，另外对高剂量组给药后0、0.5h、1h、2h的血栓形成情况进行观察。处理时间到后用两片渗透2.16 mol／L FeCl₃的滤纸敷在颈动脉外膜表面（上下各一片），记录FeCl₃使用后血压曲线变成直线的时间（即血栓形成时间）。3)血小板功能分析：尾静脉给予受试物（200μl／只），观察24h后，自下腔静脉采血5ml（3.8％柠檬酸三钠1：9抗凝），血细胞分析仪测定全血的血小板数目，分离血小板用血小板聚集仪上分析血小板的聚集情况。除此之外，我们还采集正常大鼠血液，进行了体外给药对血小板聚集率的影响实验。结果：只有10mg／kg纳米SiO₂高剂量组给药后2h和4h收缩压和舒张压与0时间点相比显著性减小（p＜0.05），各剂量组处理后各时间点心率与0时间点相比没有显著性差异（p＞0.05）。同样也只有10mg／kg纳米SiO₂组处理2h和4h后的血栓形成时间与模型组相比显著性缩短（p＜0.05）。动物给药观察24h后，10mg／kg微米SiO₂组和各剂量纳米SiO₂组与NS对照组相比，血小板数量没有显著性差异（p＞0.05），而5mg／kg和10mg／kg纳米SiO₂组均引起了大鼠血小板聚集率的显著升高，与NS组和微米SiO₂组相比有显著性差异（p＜0.05）。正常大鼠血小板体外实验显示各剂量纳米SiO₂组可促进大鼠血小板的聚集，与NS组和微米SiO₂组相比有显著性差异（p＜0.05）。研究三：纳米SiO₂对职业接触人群心血管系统的影响方法：虽然全国纳米SiO₂的生产规模较大，但生产纳米SiO₂的职业工人数量并不多，所以我们准备先进行人群探索性研究。按照预定的入选标准，我们暂时选择了一家纳米SiO₂生产规模较大厂家的纳米SiO₂作业工人9人作为职业接触组研究对象。由于人数较少，我们选取对照时进行年龄、性别、体型以及生活习惯的适当匹配，选择非纳米SiO₂接触人员9人为对照。现场调查内容包括作业环境卫生调查、健康调查和抽血检验。抽血检验项目：1)血小板相关指标测定，包括血小板计数（PLT）、平均血小板体积（MPV）、血小板压积（PCT）和血小板分布宽度（PDW）；2)凝血、抗凝和纤溶功能测定，包括血浆凝血酶原时间（PT）、活化部分凝血酶时间（APTT）、凝血酶时间（TT）、纤维蛋白原（Fib）、抗凝血酶Ⅲ（AT-Ⅲ）和D-二聚体；3)血脂及脂蛋白测定，包括总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）、血浆载脂蛋白A-Ⅰ（ApoA-Ⅰ）及载脂蛋白B（ApoB）；4)可溶性细胞间黏附分子-1（sICAM-1）和血栓调节蛋白（TM）检测；5)炎症因子测定：ELISA法测定血浆TNF-α（肿瘤坏死因子α）、IL-6（白细胞介素6）和hsCRP（高敏急性期反应蛋白）的释放水平。结果：纳米SiO₂接触组和对照组相比，PLT、MPV、PCT和PDW均没有显著性差异（p＞0.05）；凝血功能四项（PT、APTT、TT和Fib）以及AT-Ⅲ没有显著性差异（p＞0.05），接触组血浆D-二聚体显著性升高（p＜0.05）；血脂及脂蛋白指标中的TC、HDL-C、LDL-C、ApoA-Ⅰ、ApoB和ApoA-Ⅰ／ApoB均没有显著性差异（p＞0.05），只有TG一项指标明显降低（p＜0.05）；血浆sICAM-1水平存在显著性升高（p＜0.05），而两组间血浆TM以及TNF-α、IL-6和hsCRP均没有显著性差异（p＞0.05）。结论1．体外实验：纳米SiO₂具有显著的HUVECs细胞毒性。除细胞周期外，纳米SiO₂所引起的效应均比微米SiO₂严重，说明SiO₂粒径尺度的改变对其毒性有显著影响。2．动物体内实验：与体外实验结果不同，纳米SiO₂对大鼠的心血管系统的毒性较弱，只有较大剂量（中剂量或高剂量）纳米SiO₂组对大鼠的收缩压、舒张压、血栓形成以及血小板聚集率有显著影响。3．人群研究：人群探索性研究并没有发现纳米SiO₂对职业接触人群心血管系统的明显效应，只引起了血浆D-二聚体、TG及sICAM-1的显著性改变。对于纳米SiO₂是否存在远期心血管毒性效应，能否增加未来心血管事件的发生率还有待加大样本量及进行随访研究。更多还原

【Abstract】 Background and objectiveAt present, nanotechnology is developing rapidly in the world. Along with many new nanomaterials are being applied to many domains, the exposure opportunity of the researchers, the producers, the consumers, the nano-waste workers and the ecological environment to the nanomaterials increased a lot. It’s uncertainty of the safety of the nanotechnology, and then theresearches have received the widespread attention of the experts come from various countries, the related administrants and the public.Some nanomaterials could penetrate the blood-brain barrier, the blood-eye barrier and the blood-testis barrier, thus harms the the security of organism possibly, such as single-walled carbon nanotube （SWCNT） and Fe₂O₃-Glu nano-pellet. These nanomaterials may transfer to kinds of organs of the animals, such as eye, heart, liver, spleen, lung, kidney, stomach, brain, skeleton, muscle, intestine, skin, blood and germ cells. Researches have indicated the nanomaterials may harm the organisms in the level of cell, sub-cell, genome, proteome and the animal. Compares with the micro-level of homogeneous particles, the nanoparticles show a higher biological activity, and the potential toxicity may be also bigger. Considered from the system toxicology, the eyes of the researches focus on the lung toxicity at present.Lots of epidemiology researches indicated that the incidence rate and mortality of cardiovascular disease may be related with the air pollutants, and the cardiovascular disease threatens the human health seriously. Therefore, the cardiovascular effects of the nanoparticles have been the focus of public. Until now, we not yet find the report of the cardiovascular toxicity of nano-SiO₂. In view of the fact that nanomaterials mayplay an important role in the cardiovascular toxicity, this project selects the nano-SiO₂ as the research object, uses the methods of in vitro, in vivo and the population studies, then gives a more comprehensive observation of the effect of nano-SiO₂ on cardiovascular system. So, we could not only observe the effect of nano-SiO₂ on the cardiovascular system in the animal and population level, but understand the molecular mechanism of the vessel endothelial cell damage in vitro.Methods and resultsThe project will be separated into three parts as follows:Study I: The toxicity of nano-SiO₂ particles in the human umbilical vein endothelial cells （HUVECs）.Methods: Prepared the nanomaterial, then take HUVECs as the experimental object. The value of IC₅₀ was measured by the trypan blue assay. After the dosages was definited, we use the Annexin V and PI double dyeing method and cell cycle kit to observe the effects of nano-SiO₂ on cell apoptosis and cell cycle by FCM. DNA damage examination: the foci of γH2AX of each group were measured by FCM and immunofluorescence technology. Inflammation cytokines: the release of TNF-α, IL-6 and hsCRP in the culture was determined by ELISA.Results: Probit analysis was used to calculate the 24h IC50 of nano-SiO₂ in HUVECs, and the value was 194.9μg/ml approximately. In this experiment, 100μg/ml （approximately 50% IC50） used as the highest dose of nano-SiO₂. 25μg/ml, 50μg/ml and 100μg/ml nano-SiO₂ could induce the HUVECs cell apoptosis （10.5%、17.9% and 15.3%） and causes the capture of cell cycle at G1 period remarkably, All the nano-SiO₂ groups could cause significant DNA damage, the percentage of positive cells were 14.2% （25μg/ml）, 23.9% （50μg/ml） and 25.9% （100 μg/ml） alternately. All the nano-SiO₂ groups could increase the release of the inflammation cytokine TNF-α remarkably, and IL-6 was induced remarkably on 50μg/ml and 100μg/mlnano-SiO₂ besides 25μg/ml. The above results elicit the cytotoxicity of nano-SiO₂ in HUVECs. The effect which nanometer SiO₂ causes was more serious than micro-SiO₂ besides the cell cycle, it is demonstrated that the toxicity of nano-SiO₂ was mainly due to the particle nanometer size.Study II: Cardiovascular toxicity of nano-SiO₂ particles in rats Methods: The effect on mouse blood pressure and heart rate of nano-SiO₂: the left common carotid artery was separated （approximately 2 cm） after anaesthesia. Then the artery was jointed with pressure transducer, the signal of blood pressure and heart rate could be aquired by Medlab-E biology gathering system. The establishing of thrombus model: the method was carried on the improvement of the protocols using by Kura and Tang Yinghong, separated the left common carotid artery to monitor the blood pressure and heart rate as above. MedLab-E biology signal gathering processingsystem monitor blood pressure and heart rate. Placed two pieces of filter paper that was added with 20μl 2.16 mol/L FeCl₃ on the up and down face, recorded the time of the blood pressure curve turn to the straight line after the use of filter paper （namely thrombus form time）. Blood platelet functional analysis: some minutes after the intratracheal instillation of nano-SiO₂ into the lungs of rats （200μl/rat）, 5ml whole blood was collected from the under cavity vein （3.8% citric acid three sodium 1: 9）. Using the automatic blood analyzer to measure the blood platelet number, and after the separation of blood platelets, the gather situation was analyzed by the blood platelet aggregater.Results: Only the highest dosage of nano-SiO₂ （10 mg/kg） group decreased the systolic pressure and the diastolic pressure after 2 h and 4 h treatment compared with that at 0h, and heart rate had no difference between all groups. Similarly, only 1h、2h and 4h after the administration of the highest dosage nano-SiO₂ induced the formation of thrombus remarkably. After 24 h, the micro-SiO₂ group and variousdosage nano-SiO₂ group could not affect the platelet quantity compared with the NS control group, but the medium dosage and the highest dosage of nano-SiO₂ group could increased the platelet aggregation of rat remarkably.Study III: The cardiovascular toxicity of nano-SiO₂ in occupational exposure population.Methods: Chosen 9 nano-SiO₂ occupational workers from a nano-SiO₂ factory with high production. Because the population is less, we select the control workers match with age, sex, stature and the life habits. Then we also choose 9 workers who had no exposure history of nano-SiO₂. The field epidemiology includes the investigation of workplace sanitary, occupation and the exposure history, health situation and blood examination. Blood examination issues included blood platelet （PLT, MPV, PCT and PDW）, hemoglutination function, fibrinolytic function （PT, APTT, TT, Fib, AT-III and D- dimer）, blood lipids, lipoprotein mearsurement （TC, TG, HDL-C, LDL-C, ApoA-I and ApoB）, sICAM-1, TM and inflammation cytokines（TNF-α、IL-6 and hsCRP）Results: The nano-SiO₂ exposure group compared with the control group, the values of PLT, MPV, PCT and PDW has no difference; the four items of hemoglutination function （PT, APTT, TT, Fib） and AT-III also have no difference. Only the plasmas D-dimer has a significant difference between the two groups; TC, HDL-C, LDL-C, ApoA-I, ApoB and ApoA-I/ApoB does not have significant difference, only TG have a significant difference between the two groups; Plasma sICAM-1 has a significant difference, but TM and inflammation cytokines have no significant difference between the two groups.Conclusion1. In vitro: Nano-SiO₂ has the remarkable HUVECs cytotoxicity, and promotes the release inflammation factor remarkably. Besides the cell cycle, the effect which nano-SiO₂ causes is more serious than micro-SiO₂, show that the SiO₂ particle size has the remarkable influence to its toxicity.2. In vivo: Compared with result of in vitro assay, nano-SiO₂ shows a weak cardiovascular toxicity in rat. Only the higher dosage （medium or highest） of the nano-SiO₂ group could decrease the systolic pressure, the diastolic pressure, the thrombus formation, and platelet aggregation rate significantly.3. Population study: Nano-SiO₂ increases the level of plasma D-dimer and sICAM-1 significantly, and decreases the TG significantly. Maybe nano-SiO₂ have the potential remote harm effect on cardiovascular system? We could not give the answer, we should increase the sample size to confirm the effects of nano- SiO₂ in population study, and do fellow-up study in the future.更多还原