摘要
纳米二氧化钛(纳米TiO2)是一类新型的无机纳米材料，近年来被广泛应用于化妆品、服装、环境保护等领域。由于纳米TiO2具有较强的光化学活性，大量使用与人体皮肤接触，其皮肤安全性引起人们的广泛关注。目前，国内外关于纳米TiO2的毒性研究大都来自体外和短期的动物毒性实验，未见系统的纳米TiO2透皮行为及紫外光（UV）诱导下致皮肤损伤机制的研究。本论文研究了不同粒径（4～90 nm），不同晶型（锐钛矿型、金红石型、混合晶型）纳米TiO2的离体、在体透皮行为以及器官蓄积；研究UV诱导下纳米TiO2产生活性氧自由基（ROS）的途径和对人角质形成细胞（HaCaT）的损伤机制；探讨抗氧化剂N-乙酰半胱氨酸（NAC）降低纳米TiO2的氧化损伤效应。开展的主要工作如下：
（1）采用水平透皮扩散池，研究纳米TiO2离体透皮行为，以乳猪和Bulb/c裸鼠为模型，研究纳米TiO2在体透皮行为。离体透皮结果表明：不同粒径及晶型的纳米TiO2均没有透过猪耳皮肤角质层，胶带剥离实验结果提示纳米TiO2主要沉积在皮肤表面的毛孔及褶皱中。在体透皮结果表明：乳猪猪耳皮肤在体透皮30 d后，通过透射电子显微镜（TEM）及病理组织切片观察，纳米TiO2可穿透猪耳皮肤角质层，进入表皮，且可进入表皮细胞胞浆内，并对细胞产生一定程度的损伤，主要表现为细胞核周围出现空泡及细胞间隙模糊，细胞间桥粒连接消失；Bulb/c裸鼠整体透皮60 d 后，不同晶型及粒径纳米TiO2均不同程度的穿透裸鼠皮肤，进入皮下各组织和脏器中，并引起相应的组织病理学改变，主要表现为组织炎性细胞浸润及灶性坏死，裸鼠暴露皮肤出现褶皱加深、松弛、无光泽等老化现象，光镜下皮肤组织呈现表皮角化
过度、表皮变薄及深层细胞萎缩等病理学改变，皮肤组织匀浆样品中羟脯氨酸(HYP)含量、超氧化物歧化酶（SOD）水平显著降低，丙二醛（MDA）含量明显上升。试验结果提示皮肤老化及损伤均与氧化应激相关。
（2）采用噻唑蓝（MTT）法研究UV诱导下纳米TiO2的细胞毒性，采用流式细胞术、荧光分光光度法研究细胞凋亡、胞内ROS含量和线粒体膜电位（Δψm）的变化
2020端午节是几号规律， TEM观察纳米TiO2在细胞中的分布。结果表明，UV诱导下纳米TiO2具有较强的细胞毒性，能够浓度依赖性地降低HaCaT细胞的存活率，UV诱导下纳米TiO2产生大量的ROS，引起HaCaT细胞的氧化损伤、细胞凋亡及线粒体Δψm的降低。实验中选用的不同粒径、不同晶型的纳米TiO2均可穿越细胞膜进入HaCaT细胞，并主要以团聚的形式存在于细胞的胞浆中。
（3）通过加入线粒体呼吸链抑制剂，线粒体解偶联剂，线粒体通透性转换孔(PTP)抑制剂，研究UV诱导下纳米TiO2产生ROS的途径及损伤细胞的机制。结果表明，线粒体呼吸链抑制剂及氧化磷酸化解偶联剂均能明显影响纳米TiO2引起的细胞内ROS的产生；线粒体以外的ROS来源，如脂氧酶、环氧酶、NADPH氧化酶等对纳米TiO2诱导的细胞内ROS的产生没有明显影响；一氧化氮合酶催化代谢对纳米TiO2诱导的细胞内ROS的产生有一定程度的影响；PTP抑制剂环孢霉素 A(CsA)能够抑制纳米TiO2的细胞毒性。上述结果提示，线粒体呼吸链可能是纳米TiO2致细胞内ROS过量产生的主要来源，线粒体通透性转变在纳米TiO2的细胞毒性中具有重要的作用。
名字
（4）通过加入抗氧化剂NAC预处理细胞，研究UV诱导下NAC降低纳米TiO2的氧化损伤效应。结果表明，NAC使细胞凋亡率显著性下降，明显抑制细胞胞内ROS 的产生和蓄积，维持线粒体Δψm稳定，保护线粒体；用逆转录-聚合酶链反应(RT-PCR)检测HaCaT细胞角蛋白6（Keratine 6）的表达，纳米TiO2可明显抑制Keratine 6的表达，而NAC能使其表达显著上调。结果提示NAC可通过拮抗胞内氧化应激损伤而降低纳米TiO2的细胞毒性。
综上所述，本工作对纳米TiO2的皮肤安全性进行了较系统的研究。首次研究了纳米TiO2在乳猪猪耳皮肤和BALB/c 裸鼠颈部皮肤上的长时间在体透皮行为，发现经长时间的皮肤暴露，未经修饰的纳米TiO2可穿越皮肤的角质层，产生器官蓄积并对皮肤及器官造成损伤。研究了UV诱导下纳米TiO2对皮肤细胞的损伤效应及机制，结果表明UV诱导下纳米TiO2诱发细胞内ROS的过量产生，损伤细胞膜及线粒体，引起线粒体损伤，从而诱导细胞凋亡；初次阐明了线粒体呼吸链是纳米TiO2致细胞内ROS过量产生的主要来源，线粒体通透性转变在纳米TiO2的细胞毒性中具有重要香港性感女星
叶青个人资料的作用；抗氧化剂NAC可以通过拮抗胞内氧化应激损伤而降低纳米TiO2的细胞毒性。研究工作为纳米TiO2的安全应用提供实验依据。
关键词：纳米二氧化钛；透皮行为；紫外光诱导；人角质形成细胞（HaCaT细胞）；
活性氧自由基（ROS）；氧化应激；抗氧化剂
Abstract
As a new inorganic nanomaterial, nano titanium dioxide (nano-TiO2) has been widely used in cosmetic creams, costume, and environmental protection. Owing to the existence of nano-TiO2 in environment and sunscreen products, skin is inevitable to become potential targets. However, nano-TiO2 has high photochemical activity, its skin safety has attracted attention widely. At present, the investigation on the toxicities of nano-TiO2 is focued on its toxicities in vitro and animal short term exposure, while the study on nano-TiO2 skin penetration and potential skin damage under UV irradiation is limited. In the present study, we explored the penetration of nano-TiO2 in vitro and in vivo with different sizes (4～90 nm) and different crystal forms (anatase, rutile and anatase/rutile). The cytotoxicity and oxidative stress induced by nano-TiO2 under UV irradiation in human keratinocytes (HaCaT) cells were investigated. Furthermore, the potential of N-acetylcysteine (NAC) in protecting the HaCaT cells from nano-TiO2 induced toxicities was also studied. The main results are as followings:
(1) Horizontal diffusion chamber, sucking pig and Bulb/c nude mice were used to investigate skin penetration of nano-TiO2 in vitro and in vivo. The study in vitro indicated that nano-TiO2 could not penetrate through stratum corneum and mostly locate in skin pores and folds. However, study in vivo
indicated that after 30 days skin exposure of sucking pig ear, nano-TiO2 could penetrate through horny layer and locate in cytoplasm of cells. Cells damages, such as vacuole around nucleus, desmosomes disappearance were also observed. Further more, the study in mice obtained a more clearly presentation on its tissue distribution and potential toxicities after skin exposure for 60 days. The most important pathological changes were inflammatory infiltrates and foci of necrosis. The skin of Bulb/c nude mice showed more wrinkles, loose and lacking brightness with the significant changes in SOD and MDA levels, which suggested that these lesions are likely to be mediated with oxidative stress by the deposited nano-TiO2. Accordingly, the content of collagen expressed as HYP was also reduced in mice skin. It indicates that topically applied nano-TiO2 on skin with a prolonged period could induce skin aging.
(2) MTT assay was used to evaluate the cytotoxicity of nano-TiO2. Flow cytometry
and fluorospectrophotometer were used to evaluate the apoptosis, ROS level and mitochondrial membrane potential. Transmission electron microscope(TEM) was used to observe the distribution of nano-TiO2 in HaCaT cells. The results of toxicities study in vitro on HaCaT cells showed that nano-TiO2 could significantly reduce cell viability in a dose-dependent manner. The ROS generated by nano-TiO2 produced oxidative stress in HaCaT cells, the mitochondrial membrane potential of cel
ls was declined significantly while the apoptosis rate was increased. Nano-TiO2 was appeared to enter the cells by endocytosis and stay in cytoplasm as aggregates and/or agglomerates.
(3) The way of nano-TiO2 generating ROS and the cytotoxicity mechanism were also investigated by adding the inhibitors of respiratory chain and permeability transition pore, uncouplers of mitochondrial oxidative phosphorylation. Results showed that pretreatment inhibitors of mitochondrial respiratory and the uncouplers of mitochondrial oxidative phosphorylation resulted in obvious changes in the production of intracellular ROS induced by nano-TiO2. The contribution of extramitochondrial ROS source to ROS overproduction induced by nano-TiO2 were also examined. The ROS sources of lipoxygenase, cyclooxygenase and NAD(P)H oxidase had no significant effect on the ROS production induced by nano-TiO2. To some extent, NO synthase had effect on the ROS production. Cyclosporin A could reduce the cytotoxicity of nano-TiO2. These results indicated that mitochondrial respiratory chain maybe the main source of intracellular ROS production induced by nano-TiO2. The mitochondrial permeability transition plays an important role in the cytotoxicity of nano-TiO2.
(4) The potential of NAC in protecting HaCaT cells from nano-TiO2 induced oxidative stress and apoptosis under UV irradiation was also investigated. The results showed that NAC could prevent na
no-TiO2 inducing oxidative stress and apoptosis in HaCaT cells, maintain mitochondrial membrane potential and protect mitochondrion. NAC inhibited the suppression of nano-TiO2 on Keratine6 mRNA expression. These results indicated that the inhibitory mechanism of NAC on nano-TiO2 induced apoptosis involves the modulation of the intracellular ROS level and the suppression of oxidative stress.海尔冰箱评价
中国最大的省In conclusion, the safty of nano-TiO2 on skin was evaluated systematically in this paper. The skin penetration of nano-TiO2 in vivo was observed in sucking pig and Bulb/c