纳米二氧化钛在神经干细胞中的胞吞、胞排及其机理的研究

发布时间：2018-01-27 00:44

本文关键词： 纳米二氧化钛 C17.2细胞胞吞胞排生物相容性　出处：《上海大学》2013年硕士论文　论文类型：学位论文

【摘要】：随着纳米技术的迅速发展，纳米材料的广泛应用和纳米材料产量的迅速增加，人们开始关注纳米材料的安全性。例如，在生物医学领域，纳米材料作为药物传输的载体对病理组织和细胞有很好的靶向性并且能够起到安全有效的药物疗法的作用。其中，纳米二氧化钛(nano-TiO_2)因其独特的物理和化学性质在诸多领域得到广泛的应用。纳米二氧化钛对于生物体的安全性研究变得尤为重要，特别是纳米材料在细胞内的基本代谢过程，如细胞的胞吞作用和胞排作用。神经干细胞(NSCs)是神经系统中各类神经细胞的原组细胞，它既具有增值特征又拥有分化发育的潜能。目前，对于纳米TiO_2的生物毒性、环境效应、与生物组织的相互作用还没有系统的研究结果。针对目前的研究现状，本论文拟在细胞水平上，以神经干细胞为细胞模型，对三种不同形貌和粒径的纳米TiO_2的胞吞和胞排作用进行定量的研究。纳米颗粒的物理化学性质及表面性能决定其生物学作用，论文首先对三种不同粒径不同形貌的纳米TiO_2的纯度、粒径、形貌、晶型、表面电势等进行了充分的表征。随后采用CCK-8和活性氧检测等手段评估纳米TiO_2对神经干细胞的细胞活力和细胞内ROS水平的影响。毒性检测结果显示，纳米TiO_2在低浓度下对细胞无毒。纳米TiO_2对细胞的毒性存在着时间效应和浓度效应，当纳米颗粒浓度达到150mg/L以上时，细胞活力开始下降，细胞内的活性氧显著增加，但与纳米材料的尺寸和形貌没有相关性。纳米TiO_2的胞吞和胞排过程研究：胞吞作用随着时间的增加，摄取量也明显增加，在48h后摄取量几乎达到了饱和状态。粒径越小的纳米颗粒胞吞速度越快，更容易进入细胞，管状的纳米颗粒比球状的更难进入细胞。对于不同浓度的纳米TiO_2来说，纳米材料的浓度增加，细胞摄取量也成倍增加，但对摄取率没有影响。胞吞作用受纳米材料的尺寸、形貌和浓度的影响。细胞对纳米管的摄取速度比对另外两种纳米颗粒的摄取速度快。根据胞吞量随时间的变化曲线，选择48h为起点，采用细胞多步传代的方法，通过激光共聚焦显微镜对细胞的胞排过程做了动态的追踪实验。在移除细胞外的纳米颗粒后，随着时间的变化，围绕在细胞核周围的纳米颗粒由团聚慢慢分散，最后被细胞排出。ICP-AES的分析结果显示随着时间的增加，细胞内的Ti元素的浓度显著减少。浓度为50mg/L的三种纳米TiO_2在24h的胞排率分别为35.0%(NP1)，34.6%(NP2)和41.7%(NT)。当纳米TiO_2的浓度增加为100mg/L时，胞吞量增加一倍而胞排量却没有同样增加一倍。纳米材料的胞排作用受到细胞胞吞的纳米材料的数量的影响。例如，就NT而言，随着纳米TiO_2浓度的不同，胞排率从41.7%降低至26.7%。研究结果显示，细胞的胞排作用受到加入细胞的纳米材料的浓度、细胞胞吞的纳米材料的浓度和纳米材料的形貌的影响，但是与纳米材料的尺寸无关。胞吞和胞排作用的机理研究：与37°C条件相比，在4°C下，纳米TiO_2的胞吞和胞排量明显减少了，说明细胞对于纳米材料的胞吞作用和胞吞作用都存在着能量依赖，并且其胞吞过程是通过受体介导来完成的。随后，我们研究了血清对纳米TiO_2的胞吞和胞排作用的影响。结果显示，没有血清的条件下，对于三种不同的纳米TiO_2，胞吞量和胞排量都被抑制了，其中胞吞量分别被抑制了24.9%(NP1)，18.6%(NP2)和30.4%(NT)，同时，胞排量被抑制了23.0%(NP1)，，19.5%(NP2)和58.2%(NT)。在透射电镜下观察到，经过血清处理的纳米材料表面吸附了一层胶状的血清蛋白，吸附了血清蛋白的纳米颗粒更容易进入细胞。纳米颗粒-蛋白的复合体减少了纳米粒子的团聚，促进了细胞的胞吞和胞排作用。在胞排过程中，细胞形貌发生了变化，其原因是细胞微管发生了变化，经纳米材料处理过的细胞微管不规则排列。微管起着细胞内物质运输的路轨的作用，破坏微管会抑制细胞内的物质运输，这也可能是引起胞排过程中细胞内活性氧略微升高的原因。
[Abstract]:With the rapid development of nanotechnology, rapidly increasing widely the application of nano materials and nano materials production, people began to pay more attention to the safety of nano materials. For example, in the field of biomedicine, nano materials as drug delivery carrier has good targeting and to safe and effective medication effect on pathological tissue and cells. Among them, nano titanium dioxide (nano-TiO_2) because of its unique physical and chemical properties have been widely used in many fields. Nano titanium dioxide is particularly important for the safety of the organism becomes, especially the basic metabolism in the cells of nano materials, such as cell endocytosis and exocytosis of neural stem cells. (NSCs) is a group of cells of various neural cells in the nervous system, it not only has the characteristics of value-added have differentiation potential. At present, the biological toxicity of TiO_2 nanoparticles Of environmental effects, the interaction with the biological tissue has no systematic research results. According to the current situation of the research, this paper aims at the cellular level, the neural stem cells as a cell model to study on the three kinds of different morphology and particle size of nano TiO_2 endocytosis and exocytosis effect quantitatively.
The physical and chemical properties and surface properties of nanoparticles determines its biological function, purity, firstly, three different sizes of different morphologies of TiO_2 nano particle size, morphology, crystal type and surface potential were fully characterized. Then using CCK-8 and ROS detection in the assessment of the impact of nano TiO_2 on the level of ROS neural stem cells, cell viability and cell toxicity. Test results showed that nano TiO_2 with low concentration of cell toxicity. Toxicity of nano TiO_2 on cell has time effect and concentration effect, when the concentration of the nano particles can reach more than 150mg/L, the cell viability decreased, intracellular reactive oxygen species increased significantly, but the size of and there was no correlation between morphology and nano materials.
Study on nano TiO_2 endocytosis and exocytosis, endocytosis as time increases, consumption also increased significantly, after 48h intake almost reached saturation. Particles size of the endocytosis speed faster, more easily into the cells, tubular nano particles is more difficult than the ball into the cells. For different concentrations of nano TiO_2, increasing the concentration of nano materials, cell uptake also increased exponentially, but has no effect on the uptake rate of endocytosis by nano materials. The size, morphology and impact velocity on the nanotube concentration. The uptake of cells than to the other two kinds of nanoparticles uptake speed according to the. Endocytosis volume versus time curves, choose 48h as the starting point, using the method of multi step cell passage, by laser scanning confocal microscopy of cell line process to make the tracking experiment dynamic. Nanoparticles in the removal of extracellular Later, with the change of time, around the nanoparticles from agglomeration slowly dispersed around the nucleus, was finally discharged.ICP-AES analysis results showed that the cells increased with time, concentrations of Ti in cells was significantly reduced. The concentration of three kinds of nano TiO_2 50mg/L in 24h cell line rates were 35% (NP1). 34.6% (NP2) and 41.7% (NT). When the concentration of nano TiO_2 increased to 100mg/L, doubling the amount of endocytosis and cell displacement is no doubling. Nano materials are affected by the cell exocytosis endocytosis the number of nano material effects. For example, NT, with nano TiO_2 different concentration of exocytosis rate decreased from 41.7% to 26.7%. the results of the study showed that the cell line is affected by the concentration of nano material into cells, cell morphology influence endocytosis of nanomaterials and the concentration of nano materials and nano materials, but the size of Irrelevant.
Study on the mechanism of endocytosis and exocytosis effect: compared with 37 DEG C, under the temperature of 4 C nano TiO_2 endocytosis and cell volume significantly reduced, indicating that cells for nanomaterials endocytosis and endocytosis are energy dependent, and its endocytosis is mediated by the receptor to complete. Then, we study the effect of serum on nano TiO_2 endocytosis and exocytosis effect. The results showed that no serum conditions for three different kinds of nano TiO_2, endocytosis and cell displacement are suppressed, which respectively endocytosis was inhibited by 24.9% (NP1). 18.6% (NP2) and 30.4% (NT), at the same time, the displacement of cells was inhibited by 23% (NP1), 19.5% (NP2) and 58.2% (NT). Observed under transmission electron microscope, the surface of the nano material after serum treatment serum protein adsorption layer of colloid, adsorption of serum albumin nanoparticles more easy access to the nanoparticle cells. Protein complex reduced the agglomeration of nanoparticles, promote cell endocytosis and exocytosis. In the exocytosis process, changes in cell morphology, which is due to the changes of cell microtubules, nanometer material treated cells arranged irregularly. Microtubule plays an intracellular material transport track effect of disruption of microtubules can inhibit the intracellular material transportation, which may be caused by exocytosis of intracellular reactive oxygen species slightly higher.

【学位授予单位】：上海大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R318.08;TB383.1

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