【作者】 刘慧婷；

【导师】 洪法水；

【作者基本信息】 苏州大学 ， 免疫学， 2014， 博士

【摘要】 纳米二氧化钛(TiO2)是一种不易燃烧的无味白色粉末，以各种形式存在，如锐钛矿型、金红石型以及板钛矿型。该材料具有高度稳定、防腐蚀、高效光催化等特性，常被作为一种着色剂广泛应用于化妆品、医药以及涂料工业。随着纳米技术的发展，越来越多的纳米粒子被释放到环境中。其颗粒具有较大的比表面积，小尺寸效应导致纳米TiO2的生物毒性不同于常规粒子。近来，纳米粒子对人类和环境的影响已经引起科学研究者的关注。肝脏是机体主要的糖、脂肪、蛋白质、维生素和激素等物质的代谢器官，具有丰富的血液供应和储备以及独特的形态结构，而且还具有分泌、排泄和生物转化等重要功能。除此之外，肝脏在重金属解毒过程中也起着非常重要的作用。纳米TiO2对肝脏损伤的分子作用机制等问题尚未得到深入的研究。鉴于此，本文将分别以不同剂量连续腹腔注射两周，以及连续灌胃30天、60天、90天两种染毒方式研究纳米TiO2对ICR小鼠肝脏的作用，探讨引起肝脏炎症的分子机制。通过本文的研究，可从分子水平上深入地了解纳米材料的应用对人体健康可能带来的毒副作用，进一步推动纳米材料在生物医药领域的应用和发展。论文结果如下：1.对ICR小鼠进行连续两周腹腔注射纳米TiO2（5nm，0，10，50，100，150mg/kg BW）和体相TiO2（150mg/kg BW），实验结果如下：1.1在各器官中钛的累积量由高到低的顺序是肝>肾>脾>肺>脑>心脏（P <0.05），说明纳米TiO2主要积累于肝脏、肾脏和脾脏中。各器官中钛的积累与器体比的改变、器官的损伤密切相关。小鼠血清生化指标异常：中高剂量纳米TiO2(50,100,150mg/kg BW)处理后，肝碱性磷酸酶(ALP)，丙氨酸转氨酶(ALT)，亮氨酸氨基肽酶(LAP)，假性胆碱酯酶(PChE)，总蛋白(TP)和白蛋白(ALB)六项指标显著高于对照组(P <0.05或P <0.01)；随着纳米TiO2处理剂量的增加，小鼠肾脏功能相关血清生化指标肌酐(Cr)逐渐增加，血清Ca和血清P含量与对照比未达显著差异(P>0.05)， UA和BUN随处理剂量的增加逐渐下降；中高剂量处理组小鼠血清肌酸激酶(CK)，乳酸脱氢酶(LDH)，天冬氨酸转氨酶(AST)和-羟丁酸脱氢酶(HBDH)活性明显增加(P <0.05或P <0.01)；总胆固醇(TCHO)、三酰甘油(TG)，葡萄糖(GLU)，高密度脂蛋白胆固醇(HDL-C)浓度逐渐增加(P <0.05)，说明血糖及血脂代谢异常。1.2纳米TiO2可造成小鼠肝脏病理性改变，通过病理切片可以观察到炎症细胞浸润，肝细胞肿胀，空泡化，弥散性嗜酸性变，血管内有淤血。通过透射电镜可进一步观察到中高剂量处理组染色质固缩、分布不均匀，线粒体肿胀、空泡化，细胞内出现凋亡小体。1.3与同剂量微米级TiO2（10-15m）相比，纳米TiO2（5nm）引发的肝脏炎症更严重。出现的血清生化指标改变、病理和细胞亚结构变化、细胞因子表达的变化与对照组相比更明显。1.4纳米TiO2造成肝脏损伤的原因之一是体内氧化还原系统的失衡。中高剂量纳米TiO2处理后，ROS（如O2，H2O2）积累明显加快，膜脂过氧化明显加重(P <0.01)；超氧化物歧化酶（SOD），过氧化氢酶（CAT），抗坏血酸过氧化物酶（APx），谷胱甘肽过氧化物酶（GSHPx）活性受到了显著的抑制，还原型谷胱甘肽（GSH）和抗坏血酸水平、GSH/GSSG和AsAH/AsA比值随处理剂量升高而降低（P <0.05或P <0.01）。由于抗氧化酶活性受到抑制，并减慢了抗氧化剂的氧化还原循环，肝脏对于ROS的清除能力下降，导致ROS大量积累，肝脏受到氧化胁迫而发生损伤。1.5纳米TiO2不仅可以随循环系统进入动物肝脏，还可以穿过核膜结合到肝DNA上。通过一系列的光谱检测，发现Ti4+可以插入到DNA碱基对中，结合到核苷酸上造成DNA收缩而产生减色效应，二级结构发生明显的改变。纳米TiO2可以与核酸中的O原子或者P原子结合，同时可以和DNA碱基对中的N原子配对，化学键长分别为1.87和2.38。纳米TiO2与DNA的结合可改变遗传信息的传递，在高剂量组还观察到了DNA的断裂。这些都可以造成肝脏的损伤。2对ICR小鼠进行连续30天灌胃（0，62.5，125，250mg/kg BW纳米TiO2），实验结果如下：纳米颗粒可以通过血液循环到达各大主要脏器，由于全身血液均需流经肝脏，导致肝脏所受影响最为明显。随着纳米TiO2处理浓度的逐步增加，各实验组小鼠血液中的下列指标：白细胞总数（WBC）、红细胞总数（RBC）、血红蛋白（HGB）、平均红细胞血红蛋白浓度（MCHC）、血小板压积（PCT）和网织红细胞（Ret）逐渐减少，而红细胞平均体积（MCV）、平均红细胞血红蛋白含量（MCH）、血小板总数（PLT）、红细胞比积（HCT）、血小板平均体积（MPV）逐渐增加。250mg/kgBW纳米TiO2处理组以上各参数与对照组相比均存在显著性差异（P <0.05或P <0.01）。血液指标的变化显示，长时间接触纳米TiO2可能使小鼠因急性反应出现内出血、骨髓造血功能低下，最终导致贫血。PLT、MPV的增加说明纳米TiO2对小鼠凝血功能有影响。RBC、HGB的减少使血液携氧量下降，可能降低小鼠的新陈代谢以及免疫反应。自然杀伤细胞（NK）的百分比、T淋巴细胞各亚群（包括CD3、CD4、CD8）百分比、B淋巴细胞百分比下降，CD4+/CD8+比值下降；IL-2分泌减少，血清中IgM水平明显下降，说明小鼠免疫功能受到了抑制。3对ICR小鼠进行连续60天灌胃（0，5，10，50mg/kg BW纳米TiO2），实验结果如下：纳米TiO2可以活化肝脏kupffer细胞，上调细胞因子如TNF-α从而激活NF-κB信号通路。多个促炎性细胞因子表达上调，如：巨噬细胞移动抑制因子(MIF)，肿瘤坏死因子-α (TNF-α)，白细胞介素-6(IL-6)，白细胞介素-1β(IL-1β)，C反应蛋白(CRP)，白细胞介素-4(IL-4)，白细胞介素-10(IL-10)的基因和蛋白表达量。促炎-抑炎平衡被打破，引发炎症级联效应。正是通过这样的信号通路（如改变TLRs以及炎性细胞因子的表达）降低了免疫能力，造成肝脏损伤。4对ICR小鼠进行连续90天灌胃（0，10mg/kg BW纳米TiO2）后，以全基因表达谱为背景，对小鼠肝RNA进行了微阵列分析测试，结果显示：1035个基因发生显著变化，其中745个基因涉及代谢过程、细胞通讯、免疫反应、细胞周期、凋亡和转运等等关键生理过程。代谢过程中差异化表达基因最为集中，为394个，所占百分比为52.2%，其中254个基因上调，140个基因下调。脂质代谢过程差异表达基因为96个，涉及“生物氧化”，“胆固醇生物合成”，“内源性固醇”，“脂类及脂蛋白代谢”等通路。免疫系统差异表达基因共98个，59个基因上调，39个基因下调。 Cfd是“固有免疫信号”通路差异表达的基因之一，它的显著下调可使补体激活下降，固有免疫应答下调。与细胞凋亡有关的基因也出现差异表达，28个上调，9个下调。上调的Axud1可以上调Axin的表达，从而激活凋亡相关的信号通路，开启细胞凋亡的过程。综上，肝脏是纳米TiO2进入小鼠机体后积累和损伤的主要器官，作用机制包括氧化胁迫，炎症瀑布级联效应，DNA损伤，免疫功能异常和多基因共同作用等。更多还原

【Abstract】 Nano titanium dioxide (TiO2) is a noncombustible and odorless white powder,exists in different forms (such as anatase, rutile, and brookite) and is widely used in thecosmetics, pharmaceutical, and paint industries as a white pigment because of its highstability, anticorrosion, and photocatalysis. More nanoparticles (NPs) are released intothe environment with the increasing development of nanotechnology. The small size andlarge surface areas endow them with an active group or intrinsic toxicity. The impacts ofNPs on human and the environment have been concerned recently by some scientistsand organizations. Liver has a rich blood supply and unique morphological structure,which is a metabolic organ (such as sugar, fat, protein, vitamin and hormone substances).Meanwhile, liver has secretion, excretion, biotransformation and other importantfunctions. In the process of heavy metal detoxification, liver also plays a very importantrole. The toxicity effects between liver in mice and nano-anatase TiO2need to be furtherstudied. Therefore, we investigated the toxicic effects and related molecularmechanisms of liver in ICR mice by administration of nano-anatase TiO2, which wasinjected into abdominal cavity for two weeks and stomach for30,60or90consecutivedays. Our findings will be benefit to well understanding of biochemical toxicitiesinduced by NPs especially on human beings, and also arousing of nanomaterialsapplication in biomedicine area.Results are listed as follows:1. ICR mice were injected into abdominal cavity with nano-anatase TiO2(0,5,10,50,100and150mg/kg BW) and with bulk TiO2(150mg/kg) every day for14days.1.1In the experimental groups, the order of the titanium accumulation in theorgans was liver> kidney> spleen> lung> brain> heart (p <0.05), and the titaniumwas mainly accumulated in the liver, kidneys, and spleen. This phenomenon showed that the accumulation of titanium in the organs was closely related to the coefficients oforgans of mice. The serum biochemicalparameters with lower dose of nano-anataseTiO2showed little difference compared with the control mice, while with higherdose of nano-anatase TiO2, the indicators of liver function, such as alkalinephosphatase(ALP), alanine aminotransferase(ALT), leucine acid peptide,pseudocholinesterase(LAP), pseudocholinesterase (PChE), total protein (TP), albumin(ALB) level, were enhanced significantly (P <0.05or P <0.01); the indicators ofkidney function, such as uric acid (UA) and blood urea nitrogen (BUN), were decreased,wheareas creatinine (Cr) was increased(P>0.05); the activities of aspartateaminotransferase (AST), creatine kinase(CK), lactate dehydrogenase (LDH), andalpha-hydroxybutyrate dehydrogenase (-HBDH), were increased (P <0.05or P <0.01). The contents of total cholesterol (TCHO), triglycerides (TG), glucose (GLU), andhigh-density lipoprotein cholesterol (HDL-C) were significantly elevated from100to150mg/kg BW groups than the control group (P <0.05). It was concluded thatnano-anatase TiO2in higher dose had serious toxicity to the liver, kidney, andmyocardium of mice and caused inflammatory response of the liver, kidney, andmyocardium and the metabolism imbalance of blood sugar and lipid.1.2Histopathology of the liver tissue: inflammatory cell infiltration, hepatocyteswelling, vacuolization, congestion of vascellum and prominent vasodilatation,wide-bound eosinophilic and focal ischemia were observed. Ultrastructure of hepatocyte:changes like chromatin pyknosis, mitochondria swelling, vacuolization and apoptoticbody were observed in the liver tissue.1.3The toxicities of titanium in100to150mg/kg BW nano-anatase TiO2(5nm)groups were more severe than bulk TiO2(15μm), such as histopathology, ultrastructure,and alterations of cytokine expression in the liver.1.4We convinced that oxidative stress was one of the toxic mechanisms due tonano-anatase TiO2. Our data showed that over production of reactive oxygen species(ROS)(such as O2and H2O2) occurred in liver of mice with increased nano-TiO2dose,indicating that the liver underwent oxidative stress. Enhancement in malondialdehyde(MDA) levels evidenced peroxidation of liver (P <0.01). The activities of superoxide dismutase (SOD), catalase (CAT), ascorbic acid peroxidase (APx) and glutathioneperoxidase (GSHPx) were significantly inhibited (P <0.05or P <0.01). The ratios ofantioxidants (ascorbate and glutathione) in the50,100and150mg kg BW nano-TiO2groups decreased significantly (P <0.05or P <0.01). Data showed that ROSaccumulation, lipid peroxidation, and decreases of hepatic antioxidative systems had allcontributed to the oxidative damage of liver caused by nano-TiO2.1.5Nano-anatase TiO2could not only enter into the liver of mouse, but alsoaccumulate in liver DNA by inserting itself into DNA base pairs or binding to DNAnucleotide with three oxygen or nitrogen atoms and two phosphorous atoms of DNAwith the Ti–O (N) and Ti–P bond lengths of1.87and2.38, respectively, altering theconformation of DNA as well. And gel electrophoresis showed that higher dose ofnano-anatase TiO2could cause liver DNA cleavage in mice. By various spectralmethods, we demonstrated that nano-TiO2may alter the transduction of geneticinformations and induce liver DNA cleavage.2. Nano-anatase TiO2(0,62.5,125,250mg/kg BW) suspensions were given toICR mice by an intragastric administration every day for30days.We observed that white blood cells (WBC), red blood cells (RBC), haemoglobin(HGB), mean corpuscular haemoglobin concentration (MCHC), thrombocytocrit (PCT)and reticulocytes (Ret) of these nano-TiO2-treated mice decreased, while meancorpuscular volume (MCV), mean corpuscular haemoglobin (MCH), red celldistribution width (RDW), platelets (PLT), hematocrit (HCT) and mean platelet volume(MPV) increased (P <0.05or0.01). The reductions of RBC, RBC, WBC, Ret and HGBsuggested that NPs could affect red blood cell production and lead to anemia. Theincrease of PLT and MPV showed a possible effect of nano-TiO2on blood coagulation.The significant decreases of RBC count and HGB concentration caused by higher dosenano-TiO2could cause a marked decrease in O2content in the blood, which mightdecrease metabolism and immune response of mice. Our data suggested that nano-TiO2decreased the percentage of T lymphocytes (including CD3, CD4and CD8), Blymphocytes and NK lymphocytes, as well as the ratio of CD4to CD8, IL-2activity andIgM of the mouse serum. These results indicated that the damage of liver function may be involved in the reduction of immune response of mice.3. Nano-anatase TiO2(0,5,10,50mg/kg BW) suspensions were given to ICRmice by an intragastric administration every day for60days.Nano-anatase TiO2could activate kupffer cells of liver. NPs increased both mRNAand protein expression levels of several inflammatory cytokines, including nucleicfactor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), macrophage migrationinhibitory factory (MIF), interleukin-6(IL-6), IL-1β, cross-reaction protein (CRP), IL-4,and IL-10. The present study indicates that the hepatitis of mice is triggered by TiO2NPs, as evidenced by alterations of expression levels of the genes and their proteinsinvolved in the signaling pathway (such as TLRs and inflammatory cytokines), and byreduction of immune capacity.4. In this section, we used the whole-genome microarray analysis technique todetermine the gene expression profile in the livers of mice exposed to10mg/kg bodyweight TiO2NPs for90consecutive days.The results showed that more than2.3%(1035genes) of the total genes (45,000genes) were significantly changed following long-term exposure to10mg/kg TiO2NPs.Microarray data showed striking changes in the expression of745genes related tometabolic process, cell communication, immune/inflammatory response, cell cycle,apoptosis, transport, and so on. Of these394genes of metabolic process,254geneswere up-regulated and140genes were down-regulated. The number of differentexpressed genes in lipid metabolism process is96. These genes were included in manymetabolic processes, like “biological process”,“cholesterol biosynthesis”,“endogenoussterols”, and “metabolism of lipids and lipoproteins”. We analyzed the immune genesand found that98genes were significantly altered by exposing to TiO2NPs. Of these98genes,59were up-regulated and39were down-regulated. The obvious reduction in theexpression of Cfd following exposure to TiO2NPs may trigger signaling cascades toactivate inflammatory programs, like the reduction of complement activation andinhibition of innate immunology. In this section, microarray data suggested thatapproximately37genes related to appotosis were significantly changed in the TiO2NP–exposed liver. Of these37genes,28were up-regulated and9were down-regulated. The over expression of Axud1could up-regulate Axin’s expression, which mightactivate the apoptotic process.In conclusion, liver is a central organ to the accumulation and damages ofnano-anatase TiO2NPs, these damages are closely associated with oxidation stress,inflammatory cascade effect, DNA damage, immune dysfunction, and molecularmechanisms of multiple genes working together.更多还原