纳米二氧化钛颗粒穿越大鼠肺泡气血屏障的电镜研究

发布时间：2018-03-23 13:47

本文选题：纳米二氧化钛　切入点：气血屏障　出处：《浙江大学》2013年硕士论文

【摘要】：[研究背景] 纳米颗粒具有独特的物理、化学和生物特性,越来越广泛地被应用于生产生活的各个领域中。它们在这些领域的应用带来了巨大的经济效益,但同时也产生潜在的生物安全性与环境安全性等问题而引起世界范围的广泛关注。已有研究结果显示纳米颗粒可引起细胞代谢、凋亡、细胞周期、应激反应、细胞信号转导和炎症反应等一系列的毒性效应。纳米颗粒进入机体后可以转运到各器官,包括大脑和心脏等重要生命器官。转运可以是简单的物理性移位,还可以发生溶解(溶解大多数主要是物理过程,少数有化学变化)和蛋白结合等化学性过程。而多项研究结果表明纳米二氧化钛颗粒(nano-TiO2)易进入上皮和间质部位,nano-TiO2在肺灌洗液中只有20%的在巨噬细胞中可以被检出,提示大多数nano-TiO2颗粒在上皮细胞或转运到间质和循环系统,然而nano-TiO2颗粒究竟是如何被转运的,现有的研究并没有给出直接的证据。 [研究目的] 利用透射电子显微镜(TEM)观察nano-TiO2颗粒是否可以直接穿越大鼠肺气-血屏障,从而由肺进入血循环。旨在为nano-TiO2的生物安全性评价、危险度管理以及纳米材料在生物医学中的应用提供有益的资料,进一步促进纳米技术和纳米产业的健康发展。 [研究方法] 本实验采用大鼠支气管灌注实验探究nano-TiO2穿越肺气血屏障的过程,具体观察指标包括：①肺组织病理切片常规HE染色：观察肺组织结构改变及nano-TiO2颗粒在肺泡的组织大体(一般指肉眼可见的)分布；②应用透射电镜技术观察nano-TiO2颗粒在肺泡的微观分布及对肺泡超微结构的影响,特别是对肺气血屏障的作用,并结合X射线能谱仪定性nano-TiO2颗粒的存在；③利用电感耦合等离子体质谱法(ICP-MS)检测灌注后大鼠静脉血中nano-TiO2颗粒在不同时间点的含量变化。 [研究结果] 1.材料表征X-衍射和透射电镜鉴定结果：试样中锐钛矿相和金红石相的比例为80%：20%,nano-TiO2材料均呈圆形晶体颗粒,测得其平均粒径约为25nm。 2.肺组织光镜观察结果：对照组,大鼠肺泡结构正常,肺泡间隔较薄,肺泡腔干净,无明显炎症变化。Nano-TiO2灌注后2h组,肺组织无明显病理性改变,肺泡完整但呈现皱缩,少量纳米颗粒以团聚状态沉积在肺泡内表面,也有少量分布在肺泡隔。Nano-TiO2灌注后6h组,大量nano-TiO2颗粒团聚物分布在大鼠肺部的呼吸性细支气管、肺泡管、肺泡囊、肺泡和肺间质；肺组织出现明显充血、出血的病理性改变,肺泡腔内有明显的渗出和炎性细胞浸润。Nano-TiO2灌注后12h组,nano-TiO2颗粒团聚物在肺组织中的分布比6h组减少,但病理性改变更加明显,部分肺泡完整性遭到破坏,肺泡结构排列紊乱或肺实变,整个肺泡结构几乎不存在。Nano-TiO2灌注后24h组,肺泡表面少见nano-TiO2颗粒沉积,间质偶见小的团聚颗粒分布,肺泡充血消失,水肿减轻,肺泡也有所修复。 3.透射电镜观察结果：在灌注后6h,肺泡腔内出现大量nano-TiO2颗粒大团聚物和链状小团聚物,肺泡腔内游离巨噬细胞形态结构完整,但胞质中有大量无膜包被的nano-TiO2颗粒团聚物。在气血屏障处,有些nano-TiO2颗粒团聚物粘附在肺泡上皮细胞上,有些nano-TiO2颗粒大团聚物已破膜进入气血屏障的肺泡上皮细胞内,有nano-TiO2链状小团聚物跨越肺泡上皮细胞和基膜进入毛细血管内皮。在灌注后12h, nano-TiO2在肺组织中分布与6h相似,可见nano-TiO2颗粒以不同方式穿越气血屏障,进入毛细血管腔,链状结构团聚物是直接穿越气血屏障进入的,块状团聚物以破膜的形式穿越气血屏障进入。在灌注后24h, nano-TiO2颗粒大量进入肺间质,肺泡隔明显增厚,接触nano-TiO2颗粒的细胞呈现细胞损伤,凋亡及坏死,细胞中出现大量空泡,细胞核缩小,线粒体肿大,脊散乱。在有nano-TiO2颗粒的Ⅱ型肺泡上皮细胞中生成大量板层小体,细胞表面微绒毛消失。 4. ICP-MS结果：与对照组相比,nano-TiO2颗粒气管灌注组在各时间点(0、2、6、12、24和48h)全血Ti元素均高于对照组,但灌注组不同时间点间比较并没有发现明显差异。 [结论] 1)本研究实验条件下,25nm粒径的nano-TiO2颗粒支气管灌注后能够跨越气-血屏障进入血液循环,其中小链状结构团聚物是以直接穿越气血屏障进入毛细血管腔的,大块状团聚物则是以破坏气血屏障的形式进入毛细血管腔的。 2)本研究实验条件下,25nm粒径的nano-TiO2颗粒支气管灌注后可造成肺充血、肺水肿、肺泡破坏和细胞凋亡等肺损伤。
[Abstract]:[research background]
Nano particles have unique physical, chemical and biological characteristics of various fields more and more widely used in production and life. They are applied in these areas has brought enormous economic benefits, but also produce biological safety and environmental safety of potential problems and attracted worldwide attention. The existing research results showed that nano particles can cause cell metabolism, apoptosis, cell cycle, stress response, signal transduction and cell toxicity of inflammatory reaction. A series of nano particles into the body can be transported to various organs, including the brain and heart and other vital organs. Transport can be physical simple shift, can also be dissolved (most dissolved is the main physical process, there are a few chemical changes) and protein binding chemical process. And a number of research results show that the nano TiO2 particles (nano-TiO2) Easy to enter the epithelial and stromal parts, nano-TiO2 was detected in lung lavage fluid in only 20% in macrophages, suggesting that most of the nano-TiO2 particles in epithelial cells or transported to the interstitial and circulatory system, but how was nano-TiO2 particle transport, the existing research does not give direct evidence.
[research purposes]
Using transmission electron microscopy (TEM) to observe whether the nano-TiO2 particles can be directly through the rat lung qi - blood barrier and enter blood circulation by the lung. Aimed at the biological safety evaluation of nano-TiO2, provide useful information to application of risk management as well as the nano materials in the biomedical field, to further promote the healthy development of nanotechnology and nano industry.
[research methods]
This experiment adopts the process of experimental inquiry nano-TiO2 through pulmonary bronchial perfusion blood barrier in rats, including specific outcome measures: lung tissue was stained by HE, observe the lung tissue structure changes and nano-TiO2 particles in the alveolar tissue specimens (usually visible) distribution; II nano-TiO2 particles in the micro distribution of alveoli and influence on the alveoli ultrastructure was observed by TEM, especially on pulmonary blood barrier function, and combined with X ray spectrometer qualitative nano-TiO2 particles; using inductively coupled plasma mass spectrometry (ICP-MS) detection of nano-TiO2 particles after reperfusion in rat blood content changes at different time points.
[results]
1. material characterization X- diffraction and transmission electron microscopy identification results: the ratio of anatase and rutile in the samples is 80%:20%, and nano-TiO2 materials are round crystal particles, and the average particle size is about 25nm..
Results 2. lung tissue under light microscope: the control group, the rats with normal alveolar structure, alveolar septum is thinner, the alveolar cavity clean, no obvious inflammatory changes in.Nano-TiO2 after reperfusion in 2H group, the lung tissue had no obvious pathological changes, but showed complete alveolar collapse, a small amount of nano particles to agglomerate state of deposition in the alveolar surface, also a small amount of the distribution in the alveolar septum.Nano-TiO2 after reperfusion in 6h group, a large number of nano-TiO2 aggregates distribution in rat lung respiratory bronchioles, alveolar ducts, alveolar sac, alveolar and interstitial lung; lung tissue pathological changes appeared obvious hyperemia, hemorrhage, exudation and inflammatory cell infiltration was.Nano-TiO2 after reperfusion in 12h group the alveolar cavity nano-TiO2 particles distribution in lung tissues than in the 6h group decreased, but the pathological changes were more obvious, some alveolar integrity destruction of alveolar structure disorder or consolidation of the lung, the alveoli There was almost no.Nano-TiO2 perfusion in the 24h group. There was little nano-TiO2 particle deposition on the alveolar surface. Occasionally, there were small aggregation particles in the interstitium, alveolar hyperemia disappeared, edema reduced and alveoli repaired.
3. transmission electron microscope: in 6h after reperfusion, the alveolar cavities appear a large number of large nano-TiO2 particles agglomerate and a chain of small agglomerates, alveolar macrophage free intact structure, but there are a lot of cytoplasmic membrane coated nano-TiO2 particles were agglomerated. In the blood barrier, some nano-TiO2 particles agglomerate adhesion in alveolar epithelium nano-TiO2 cells, some large particles agglomerate has broken blood barrier membrane into the alveolar epithelial cells, nano-TiO2 chain small agglomerates across the alveolar epithelial cells and capillary endothelial basement membrane into. In 12h after reperfusion, nano-TiO2 in lung tissue distribution is similar with 6h, visible nano-TiO2 particles in different ways through the blood barrier, into the capillaries cavity chain structure is agglomerate directly through the barrier into blood, massive agglomerates to break the membrane form through blood perfusion in the barrier to enter the 24h, A large number of nano-TiO2 particles into the interstitial lung, alveolar septum thickening, contact nano-TiO2 granule cells showed cell damage, apoptosis and necrosis, vacuolization, cell nucleus reduced, mitochondrial swelling, ridge scattered. Generate a large number of lamellar bodies in nano-TiO2 particles of type II alveolar epithelial cells, cell surface microvilli disappeared.
4. ICP-MS results: compared with the control group, the Ti elements in the whole blood of nano-TiO2 granule tracheal perfusion group at each time point (0,2,6,12,24 and 48h) were all higher than those in the control group, but no significant difference was found in the perfusion group at different time points.
[Conclusion]
1) the experimental conditions, 25nm nano-TiO2 particle size after bronchial perfusion can cross the blood air barrier into blood circulation, the chain like structure is to agglomerate directly through the blood capillary barrier into the cavity, the bulk agglomerate is into the capillary lumen to destroy blood barrier form.
2) under the condition of this study, the 25nm particle size of nano-TiO2 particles can cause pulmonary hyperemia, pulmonary edema, alveolar destruction and cell apoptosis.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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