【作者】 李阳；

【导师】 孙志伟；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2013， 博士

【摘要】 随着纳米二氧化硅职业暴露、环境暴露及医源性暴露机会的不断增加，其对人群健康及环境的潜在危害和毒理学安全性评价逐渐受到了科学界的广泛关注。前期研究表明，纳米二氧化硅颗粒具有细胞毒性，可以诱发ROS、引起细胞氧化应激，造成细胞膜、线粒体及DNA损伤，影响细胞周期进程并诱导细胞凋亡。纳米二氧化硅颗粒致细胞多核作用是本研究组在前期工作中观察到的重要实验现象。颗粒是如何导致细胞多核形成的？形成的多核细胞最终的结局是什么？是否能够引起细胞异常增殖及更为严重的生物学后果？以上几点都是目前尚未明确并亟待回答的问题。因此，本研究首先在体外、体内条件下，对纳米二氧化硅颗粒致细胞多核的形成方式及其相关机制进行了系统探讨，以期为颗粒致多核作用的深入研究提供理论及实验依据。1.纳米二氧化硅颗粒的表征及分散稳定性研究中使用的两种无定形纳米二氧化硅颗粒（Nano-Si64及Nano-Si46）纯度均在99.9%以上，粒径分别为63.88±10.35nm及46.15±5.53nm。透射电镜及Zeta电位粒度分析仪的检测结果表明，两种纳米二氧化硅颗粒呈椭球形，大小均一，在纯水及1640培养基中均能够保持良好的稳定性，并未发生聚集。2.纳米二氧化硅颗粒剂量依赖及粒径相关的细胞毒性作用体外实验以人正常肝细胞（L-02）作为受试细胞系，首先对Nano-Si64及Nano-Si46的细胞毒性作用进行检测。分别采用CCK-8试剂盒及台酚蓝染色法检测浓度梯度为0、1、2、5、10、20、50、100、200μg/mL的两种纳米二氧化硅颗粒作用于L-02细胞24h后对细胞存活率的影响。两种检测方法得到的细胞存活率趋势基本一致，结果表明纳米二氧化硅颗粒对L-02细胞存在剂量依赖及粒径相关的细胞毒性作用，即颗粒的给药剂量越高其对L-02细胞的生长抑制作用越强，粒径较小的纳米二氧化硅颗粒表现出了更强的细胞毒性作用。3.纳米二氧化硅颗粒的细胞摄取及分布10、20、50μg/mL的两种纳米二氧化硅颗粒作用于L-02细胞24h后，分别采用FCM及ICP-AES检测细胞内颗粒度及硅含量的变化。结果表明，随作用剂量的升高，进入细胞内部的二氧化硅颗粒数目逐渐增加，同时细胞内可检测到的硅含量也是在不断升高的，并且粒径较小的颗粒更容易进入细胞内部。进一步采用TEM对纳米二氧化硅颗粒的细胞摄取及分布进行检测。结果表明，两种纳米二氧化硅颗粒均可通过细胞内吞及损伤细胞膜的方式进入细胞内部，并且既可散在或成簇的分布于细胞质中，也可沉积于溶酶体、线粒体等细胞器中。4.纳米二氧化硅颗粒对细胞形态及超微结构的影响10、20、50μg/mL的两种纳米二氧化硅颗粒作用于L-02细胞24h后，首先使用倒置相差显微镜观察细胞的生长情况及Giemsa染色后细胞的形态变化。结果表明，随作用剂量的增加，两种纳米二氧化硅颗粒对细胞形态结构的影响逐渐明显，可见变小、变圆的细胞逐渐增加，贴壁细胞逐渐减少，高剂量组还可观察到细胞核内染色质聚集及细胞死亡等现象。进一步通过SEM及TEM对L-02细胞的超微结构进行观察，可见纳米二氧化硅颗粒粘附于细胞表面，并引起微绒毛的数量减少及结构断裂；进入细胞后，颗粒能够引起细胞内线粒体、粗面内质网及溶酶体等多种亚细胞结构的异常改变。5.纳米二氧化硅颗粒对细胞的氧化损伤作用10、20、50μg/mL的两种纳米二氧化硅颗粒作用于L-02细胞24h后，细胞内ROS的检测结果表明，两种颗粒均可引起细胞内ROS水平的升高，并且存在剂量依赖及粒径相关效应。进一步对细胞内抗氧化酶SOD、GSH-PX的活性及脂质过氧化产物MDA的含量进行检测，结果表明两种纳米二氧化硅颗粒随作用剂量的升高，均可使L-02细胞内的氧化压力不断上升，颗粒对细胞的氧化损伤程度逐渐增强，并且小粒径颗粒对细胞的氧化损伤作用更加明显。6.体外条件下纳米二氧化硅颗粒的致多核作用及其相关机制10、20、50μg/mL的两种纳米二氧化硅颗粒作用于L-02细胞24h后，对双核及多核细胞发生率的统计结果表明，二氧化硅颗粒的致多核作用同样存在剂量依赖及粒径相关效应，随作用剂量的增加，实验组内双核及多核细胞的数量明显增加，并且Nano-Si46的致多核作用明显强于Nano-Si64。在细胞融合的检测中，对培养液中LDH活性及细胞内Ca+浓度的检测结果表明，两种纳米二氧化硅颗粒均可引起L-02细胞膜的损伤及细胞内Ca+浓度的升高，并且存在剂量依赖及粒径相关效应。细胞膜的损伤及Ca+浓度的升高为细胞融合的发生提供了可能，但采用实时倒置相差显微镜及激光共聚焦显微镜进行多次观察后，并未发现有两个相邻细胞通过膜的相互连接融合成一个细胞的现象。在细胞有丝分裂异常的检测中，分别采用Actin tracker green、Tubulin trackergreen及Hoechst33258对细胞骨架成分中的微丝、微管及细胞核进行标记，荧光显微镜及激光共聚焦显微镜的观察结果表明，两种纳米二氧化硅颗粒作用于L-02细胞24h后，均能够引起细胞内微丝及微管数目的降低及其在细胞内分布位置的改变；在L-02细胞进行有丝分裂的过程中，可观察到纳米二氧化硅颗粒引起了中期染色体排列的异常、后期染色体分离的异常以及末期胞质分裂的异常，进而导致细胞有丝分裂过程的失败，形成一个双核或多核细胞。另外，细胞有丝分裂过是一个耗能过程，细胞内ATP能量供应不足也可能是细胞有丝分裂失败的原因之一。研究中进一步通过JC-1荧光探针对L-02细胞内的线粒体膜电位进行检测。结果表明，两种纳米二氧化硅颗粒均能够引起细胞内线粒体膜电位的降低，并且膜电位降低的趋势与多核发生率的趋势相符。细胞核内DNA的损伤及其引起的细胞增殖障碍和G2/M周期阻滞可能是细胞多核形成的又一重要因素。采用流式细胞术对细胞增殖及细胞周期进行检测，结果表明两种纳米二氧化硅颗粒作用于L-02细胞24h后，的确引起了细胞增殖障碍及G2/M期阻滞。Western blot对细胞内增殖及周期相关蛋白含量的检测结果表明，与增殖相关的p-MEK1/2及p-ERK1/2的蛋白含量随颗粒作用剂量的增加而不断降低，与G2/M及有丝分裂相关的Cdc25C、cyclin B1、Cdc2及Cdc20的蛋白含量也是随纳米二氧化硅颗粒作用剂量的增加而逐渐降低。综上所述，纳米二氧化硅颗粒作用于L-02细胞后，可因其自身的表面活性或诱导产生ROS，引起细胞内DNA、线粒体及骨架结构的损伤，细胞增殖及周期相关蛋白含量的降低，进而通过细胞有丝分裂异常、细胞增殖障碍及G2/M期阻滞，而导致细胞多核的形成。7.体内条件下纳米二氧化硅颗粒的肝损伤作用及致多核作用使用64nm二氧化硅颗粒对ICR小鼠进行尾静脉注射，通过Dixon上下法计算其LD50估计值为262.45±33.78mg/kg。急性毒性研究以LD50估计值为基础等差向下选择了高、中、低三个剂量，即177.5、103.5及29.5mg/kg，经小鼠尾静脉注射后观察14d。亚急性毒性研究以20mg/kg作为实验剂量，连续5次注射后累积剂量为100mg/kg，观察时间分别为2w、4w及8w。病理组织学检测中观察到小鼠肝脏出现了以炎细胞浸润及肉芽肿形成为主的病理改变。急性毒性实验中，小鼠肝脏肉芽肿的数量及面积与纳米二氧化硅颗粒的剂量有关，随颗粒作用剂量的升高，小鼠肝脏病理变化逐渐明显。亚急性毒性实验中，给药后4w小鼠肝脏中形成肉芽肿的数量最多、面积最大，至给药后8w肉芽肿的数量及面积均有所减少。对小鼠肝脏实质双核及多核细胞的统计结果表明，纳米二氧化硅颗粒急性或亚急性给药后，并未引起肝脏多核细胞数目的增加，并且在急性毒性实验的高剂量组还观察到了多核细胞数目的降低，亚急性毒性实验中观察到多核细胞数目随时间延长而逐渐降低的现象，这可能与颗粒对小鼠肝脏的损伤作用有关。进一步对纳米二氧化硅颗粒引起肝脏损伤作用的机制进行初步研究。采用免疫组化的方法对小鼠肝脏切片中细胞内的DNA氧化损伤进行检测，结果表明颗粒经小鼠尾静脉到达肝脏后，并未对肝实质及肉芽肿区域细胞的DNA产生氧化损伤作用。但细胞凋亡的检测中观察到，急性毒性实验中，高剂量组可引起肉芽肿区域细胞凋亡的发生，亚急性毒性实验中，4w时肉芽肿内凋亡细胞数目较多，至8w时已观察不到细胞凋亡的阳性染色。综合上述体内实验的研究结果，纳米二氧化硅颗粒通过ICR小鼠尾静脉急性或亚急性给药后，均可对小鼠肝脏产生损伤作用，引起炎性细胞浸润及肉芽肿的产生。急性毒性实验中，随颗粒作用剂量的不断升高，肝脏肉芽肿的数目及面逐渐增加，而肝实质多核细胞数目逐渐减少；亚急性毒性实验中，肝脏肉芽肿在4w时表现的最为明显，继续观察至8w时，肉芽肿已处于逐渐恢复的阶段，而随着观察时间的不断延长，肝实质多核细胞数目是在逐渐降低的，因此，不排除肝实质细胞损伤逐渐严重的可能性。更多还原

【Abstract】 The widespread application of silica nanoparticles creates various sources forpotential human exposure. It is possible for silica nanoparticles to enter human bodythrough various means. Therefore, information focuses on the safety and hazards ofsilica nanoparticles are urgently needed. In our previous study, we have found the invitro and in vivo toxic effect of silica nanoparticles, and reported that silicananoparticles could induce multinucleation in HepG2cells for the first time. Whilethe formation and the ending of multinucleated cells are still unknown. Therefore, thepurpose of this study is to clearify the multinucleation effect and relative mechanismsof amorphous silica nanoparticles.1. Characterization of silica nanoparticlesTwo sizes of amorphous silica nanoparticles (Nano-Si64and Nano-Si46) wereinvestigated in our study. The TEM images showed that silica nanoparticles weremostly spherical with uniform size and well dispersed. The average sizes of these twoparticles are63.88±10.35nm and46.15±5.53nm, respectively. The result ofICP-AES manifested that the purity of both Nano-Si64and Nano-Si46is higher than99.9%. Dynamic light scattering (DLS) technique and zeta electric potentialgranulometer were employed to detect the hydrodynamic sizes and zeta potential ofthe two silica nanoparticles in different dispersion medium. Result showed that twoparticles possessed uniform hydrodynamic sizes and well dispersibility.2. Dose dependent and size related cytotoxicity of silica nanoparticlesTo investigate the potential cytotoxicity of the silica nanoparticles, cell viabilitywas detected after L-02cells were treated with two sizes of silica particles (Nano-Si64and Nano-Si46) at concentrations of1-200μg/mL for24h. Both two methods, cellcounting kit (CCK-8) and trypan blue staining, showed substantially the same trend,the cytotoxicity of the silica nanoparticles was dose dependent and size related, thecell viability of L-02cells decreased with the particle dose increased, and smaller silica nanoparticle exhibited stronger toxic effects.3. Cellular uptake and intracellular distribution of silica nanoparticlesFCM and ICP-AES were used to detect the Si content of L-02cells afterexposure to10、20、50μg/mL of two silica nanoparticles. Results indicated that the Sicontent elevated with the silica dose increased, and smaller particle entered the cellsmore easily. The cellular uptake and intracellular distribution of silica nanoparticleswere observed by transmission electron microscope (TEM). TEM images showed thatsilica nanoparticles could enter L-02cells through endocytosis or penetrating the cellmembrane directly; Particles dispersed in cytoplasm with individual or cluster form,and some were found in lysosomal and mitochondria.4. Morphological change of L-02cells induced by silica nanoparticlesMorphological change was detected after L-02cells were treated with10、20、50μg/mL of two silica nanoparticles24h. Morphological observations under phasecontrast microscope showd that, cells of negative control group were mostlypolygonal with little particles in cytoplasm. But in the silica nanoparticle treatedgroup, with the increase of silica dose, the morphological change of L-02cellsbecame more and more significant. Photograph of Giems staining showed that cells intreated group exhibited decrease of cell number decrease and disappearece of cellconnection; In50μg/mL silica treated group, chromatin margination and cell deathcould also be observed. Sccaning electron microscope (SEM) images manifested thatsilica nanoparticles induced cell membrane microvilli rupture and cell membranedamage. TEM observation indicated that both two silica nanoparticles could result inthe injury of many subcellular structures, such as Mitochondria, lysosomes andendoplasmic reticulum.5. Oxidative damage of L-02cells induced by silica nanoparticlesThe result of FCM showed that intracellular ROS of10、20、50μg/mL treatedgroups increased following the silica dose increasing after treated with silicananoparticles for24h. Colorimetry was introduced to detect the activity of SOD,GSH-PX and the content of MDA. Results indecated that with the silica doseincreased, the oxidative stress of L-02cells elevated obviously, and the smallar silicananoparticles exhibited higher oxidative damage effect. 6. Muitinucleated effect of silica nanoparticles and relative mechanismsThe rate of multinucleated cells was calculated after L-02cells were treated with10、20、50μg/mL of two silica nanoparticles24h. Results showed that themultinucleated effect of silica nanoparticles was also in a dose dependent and sizerelated way, that is the rate of multinucleated cells increased significantly as silicadose increased, and smaller silica nanoparticles induced more multinucleated cells.In the observation of cell fusion, real-time inverted phase contrast microscopeand laser scanning confocal microscope were used to detect cells with or with outfluorescently labeled. However, we haven’t found the phenomenon of cell fusion.In the observation of abnormal mitosis of L-02cells, Actin tracker green,Tubulintracker green and Hoechst33258were used to lable microfilament, microtubules andnucleus. Results of real-time inverted phase contrast microscope and laser scanningconfocal microscope showed that both two silica nanoparticles could induce abnormalchromosome alignment in mitosis metaphase, abnormal chromosome segregation inmitosis anaphase and abnormal cytokinesis in mitosis telophase.DNA damage induced by silica nanoparticles could lead to cell proliferationdisorders G2/M phase arrest, these might also result in the formation of multicleatedcells. Thus, we detected the content of relative regulation proteins in cell proliferationand cell cycle. Results of western blot showed that the content of p-MEK1/2andp-ERK1/2, proteins associated with cell proliferation, decreased with silica doseincreased; the content of Cdc25C, cyclin B1, Cdc2and Cdc20, proteins associatedwith G2/M cell cycle and mitosis, decreased with silica dose increased. Therefore,leading to cell proliferation disorders G2/M phase arrest could be one of the reasonsof the multinucleated effect of silica nanoparticles.7. Injury of liver and muitinucleated effects produced by silica nanoparticles inICR miceTo obtain the LD50of64nm SNPs in ICR mice, doses and interval weredesigned according to the Dixon up-and-down method. The LD50of64nm silicananoparticles was calculated using the formula provided by Dixon’s up-and-downmethod, and the entire estimate for the LD50=262.45±33.78mg/kg. For acutetoxicity study of silica nanoparticles,0,29.5,103.5and177.5mg/kg were chooseedas experimental dose, and14d was choosed as observation period. For sub-acute toxicity study of silica nanoparticles,20mg/kg was chooseed as experimental dose,after five consecutive injections, the cumulative dose was100mg/kg,2w,4w and8w were choosed as observation period.Images of pathological examination showed that lymphocytic infiltration andgranuloma formation appeared in the liver of ICR mice after silica naoparticles treatedacutely and subacutely. In the acute toxicity study, the numbers and sizes ofgranulomas in liver increased in a dose-dependent way. While the sub-acute toxicitystudy, the numbers and sizes of granulomas reached a peak in4w, and obviouslydecreased in8w.After ICR mice treated with silica nanoparticles acutely and subacutely, the rateof multinucleation of hepatocytes was calculated. Results showed that silicanaonoparticles did not increased the number of multinucleated cells in liver. Contrary,in the acute toxicity study, the number of multinucleated cells decreased significantlyin177.5mg/kg treated group; in the acute toxicity study, the number ofmultinucleated cells declined gradually. This phenomenon might be related to theliver injury induced by silica nanoparticles.In further study, we used imunohistochemistry to preliminary investigated themechanism of liver injury. Results showed that silica nanoparticles did not lead to theDNA damage of cells in both Liver parenchyma and granuloma. However, apoptosisof cells in granuloma was observed in the177.5mg/kg treated group of acute toxicitystudy and in2w and4w observation periods.The findings of the above in vivo experiments indicated that acutely andsubacutely exposure to silica nanoparticles through intravenous administration couldlead to lymphocytic infiltration and granuloma formation in the liver of ICR mice. Inthe acute toxicity study, the numbers and sizes of granulomas in liver increased in adose-dependent way. In the sub-acute toxicity study, the most serous granulomas wasobserved in4w, and has been restored in8w, but this not indicated the injury of liverwas recoverd.更多还原