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题名: 碳纳米材料诱发炎症反应并导致机体损伤的毒性通路研究
作者: 马娟
学位类别: 博士
答辩日期: 2016-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 刘思金
关键词: 碳纳米材料,巨噬细胞,促炎症反应,间接效应,尺寸效应 ; Carbon nanomaterial, Macrophages, Pro-inflammatory response, indirect effects, Size effect
其他题名: The molecular mechanisms and adverse outcome pathways underlying carbon nanomaterials induced pro-inflammatory responses and related indirect effects
学位专业: 环境科学
中文摘要:      随着纳米产业的发展,纳米材料在原材料加工、消费品生成及消费品使用的过程中,不可避免的会进入到环境中,并可能造成人群暴露。碳纳米材料,如一维的碳纳米管、二维的石墨烯、三维的富勒烯,以及其它衍生物,因优良的物理化学性质,在生产、生活中得到广泛应用,尤其是在生物医疗领域的应用潜能及优越性日益突出。因此,探究碳纳米材料生物安全性及潜在的环境及健康风险也越来越重要。尽管目前关于碳纳米管生物效应的研究很多,但是大部分工作集中在探究碳纳米材料暴露后对暴露部位细胞、组织、器官的直接毒性效应,很少有研究工作关注伴随着炎症反应状态改变产生的继发性或者代偿性效应。对于碳纳米材料暴露诱发的“有害结局路径”也称为“毒性通路”(Adverse outcome pathways,AOP)并没有明确的认识,实用的针对碳纳米材料风险评估的框架并没有建立。
    机体的免疫系统是监控、清除入侵机体的外源物质的第一道防线,免疫状态的改变会影响机体其他代谢过程及内稳态,探究纳米材料的免疫毒性机制,识别机体以免疫状态改变为起始事件的毒性通路,及其相关的间接性及代偿性效应对于更全面、深入的理解纳米材料组织、细胞相容性,揭示其生物安全性具有重要意义。探究材料本身理化性质对于生物相容性及毒性效应的影响,对于进一步基于构效关系进行纳米材料的修饰、改性,拓展其在生物医药以及环境污染控制方面的应用也具有一定的指导意义。
    本论文主要探讨碳纳米管暴露诱发周身炎症反应的间接毒性效应及氧化石墨烯活化炎症反应的理化性质决定机制。采用了原始态(P-MWCNTs),氨基化(MWCNTs -NH2)、聚乙二醇化(MWCNTs-PEG)、聚醚酰亚胺化(MWCNTs-PEI)和羧基化(MWCNTs-COOH)多壁碳纳米管模拟真实环境中的人群暴露方式及暴露剂量进行实验研究。我们发现,原始态及功能化修饰的碳纳米管(CNTs)均能诱发促炎症反应并不同程度的改变机体的铁代谢稳态。表现为:不管是什么修饰类型的  CNTs并以何种方式暴露(腹腔暴露或者肺暴露),均能检测到实验小鼠发生明显的炎症反应及机体铁代谢紊乱。从整体上考虑表面修饰对这些生物效应的影响,我们发现原始态的碳纳米管对炎症反应状态及铁稳态的扰动作用最强,而 PEG和   COOH的引入会部分减弱这种效应,而NH2则可以进一步加剧CNTs的促炎症反应。进一步的机制研究表明:碳纳米管能显著活化炎性细胞(如巨噬细胞)发生促炎症反应,并在暴露部位招募炎性细胞促进炎症反应的级联放大,导致以肝脏 hepcidin为核心的铁代谢紊乱,红细胞的合成发生障碍,脾脏内造血的代偿性增加,最终导致炎症性贫血的发生。与此同时,我们还发现碳纳米管暴露后对机体远端组织-关节存在潜在的健康风险。选择相较于其他修饰类型诱发炎症反应较为温和的羧基化碳纳米管( MWCNTs-COOH)进行机制探索发现:经碳纳米管暴露后小鼠关节部位出现以滑膜增厚、炎性细胞浸润为特征的滑膜炎症反应。进一步的机制探究说明:MWCNTs-COOH能直接活化巨噬细胞表达促炎症因子,利用巨噬细胞炎性上清孵育滑膜/软骨细胞后,可明显检测到与软骨降解相关的基质金属蛋白酶(MMPs)以及与炎症反应相关的环氧合酶(COXs)的表达量及活性增加。将 MWCNTs-COOH暴露巨噬细胞得到的炎性上清进行抗体中和后再暴露细胞发现其 MMPs及COXs的表达量降低。说明碳纳米管暴露可能使得机体的免疫敏感性增加,患免疫相关疾病的风险增加。
    氧化石墨烯(GO)因其独特的理化性质(表面积大,稳定性高)以及在水溶液中的亲水性及可分散性,在生物医药领域得到广泛应用。但是,由于 GO的理化性质差异较大,对于其生物相容性及生物安全性的认识充满争议。对材料自身的理化性质,如水平尺寸,在 GO的生物学效应及毒性效应中的影响尚不清楚。在这部分工作中,我们重点关注 GO水平尺寸对其生物效应的影响。我们利用相同的原始材料,制备得到以水平尺寸为单一变量的一组 GO样品,模拟真实环境中人群接触碳纳米材料的途径(呼吸暴露、经腹腔的内暴露及经尾静脉的血液暴露)进行体内实验,我们发现,不同尺寸 GO在激活巨噬细胞和诱发局部与系统性炎症反应方面存在很大差异:与小尺寸的 GO相比,大尺寸的  GO更易活化巨噬细胞并促发炎症反应。一系列的细胞与分子生物学研究表明:相较于尺寸较小的 GO,大尺寸材料更倾向结合于巨噬细胞的细胞膜表面,与膜的相互作用更强。
    进一步的机制研究揭示,不同大小的  GO在不同程度上激活位于细胞膜表面的Toll样受体,并差异活化其介导的下游NF-κB炎症反应信号通路。同时发现大尺寸的 GO显著刺激巨噬细胞向M1亚型极化,放大炎性级联反应。这些发现对于揭示纳米材料的潜在毒性效应,深入理解纳米材料的健康风险及生物安全性具有重要价值。
英文摘要:     With  the   development  of   Nano-technology  Industrialization,   the  expanding releasing of nanomaterial from the process of raw materials, the production of consumer goods  and usage  of commercial  products  will certainly  increase  the opportunity  of human exposure,  and this  has raised  general concerns  on their  safety. Owing  to the novel   physicochemical    properties,   carbon-based    nanomaterials   such    as   one-dimensional carbon nanotubes, two-dimensional graphene, three-dimensional fullerene and their derivatives have continuously attracted great attention in a wide range fields,especially in  biomedical and pharmaceutical  applications. Meanwhile, the  increasing use of carbon nanomaterial may cause adverse health effect through ingestion, injection and inhalation. Despite a wide spectrum of reported toxicities for carbon nanomaterial,most of them studied the toxic effects to the directly targeted tissues/organs that provide little insight  into their indirect  effects beyond their  primary targets. And there  is few research about adverse  outcome (AOP) for nanomaterials with  high exposure risk for human  beings  and a  validated  framework  for  human  health  risk assessment  upon nanomaterials has not been developed yet. The inflammatory system is the first line of surveillance,   clearance,   defense   at   the   portal-of-entry   against   foreign    agents.
    Inflammation disrupts various pathways  responsible for diverse homeostasis and may cause diverse detrimental health problems, such as anemia of inflammation and arthritis. Therefore, to investigate the mechaims about immunotoxicity and to identify AOP with the  activation   of  inflammatory  pathways   as  initiating  events   and  inflammation-associated metabolic disorders and secondary effects are of great importance for deeply understanding   the  biosafety   of  nanomaterials.   To   identify  the   physicochemical properties responsible for  the biocompability and toxicology  of specific nanomaterial is   meanful   to   further   modification   according   to   developed   structure-function relationship and  to extend the  applications in biomedicine and  contamination control field.
    In  this study,  we  endeavor  to verify  the  indirect effects  and  adverse  outcome pathways related to carbon nanomaterials induced alternation of systemic inflammatory status,  and  to   illuminate  the  molecular   mechanisms  and  size  effect   relationship underlying graphene oxide-induced pro-inflammatory effects.Apanel of CNTs were prepared including pristine multi-wall CNTs (P-MWCNTs),aminated (MWCNTs-NH2), polyethylene glycol (MWCNTs-PEG),  polyethyleneimine (MWCNTs-PEI) and  carboxylated  MWCNTs (MWCNTs-COOH).  We demonstrated that regardless of  the type of CNTs  and the exposure way (either  peritoneal cavity or lung  exposure),  significant   inflammation  and  alteration  of   iron  parameters  were observed  in mice.  The mechanisms  of  toxicity involved  enhanced  hepatic hepcidin mRNAexpression likely as a result of increased inflammatory cytokine IL-6 production,which   resulted   in  disordered   iron   metabolism   impacting   RBC   formation  and consequently the occurrence  of anemia of inflammation  in mice. Further mechanistic investigations  showed that  CNTs  activate  macrophages and  macrophage-associated pro-inflammatory responses  (including secretion  of  pro-inflammatory cytokines  and recruitment of inflammatory leukocytes), which  at least partially account for hepcidin induction,  iron  sequestration for  erythropoiesis,  anemia  and  finally  extramedullary erythropoiesis.
    Compared to  other modified  CNTs, carboxylated  multi-wall CNTs  (MWCNTs-COOH) induced milder  systemic inflammatory response in  some extent. We selected MWCNTs-COOH  in  the  following   experiment  and  found  that  MWCNTs-COOH administration  led  to  synovial  inflammation  within   knee  joints,  as  evidenced  by infiltration of pro-inflammatory  cells in synovium and  meniscus. Mechanistic studies manifested that  MWCNTs-COOH stimulated  pro-inflammatory effects  by activating macrophages, and  the secreted  pro-inflammatory  cytokines in  the supernatant  could prime the  synoviocytes and chondrocytes,  leading to enhanced  production of  a large array  of   enzymes  involved  in   articular  cartilage   degeneration,  including   matrix metalloproteinase   (MMP)   members   and   cyclooxygenase   (COX)   members  and increased enzymatic activity of  MMPs were demonstrated. Blockade of the  cytokines by antibodies significantly attenuated the production of these enzymes. This meant that exposure  to carbon  nanotubes  may promote  the  immune sensitization  of  body  and improve the risk of suffering from immunological disease.
    Graphene oxide  (GO) is increasingly used  in biomedical applications  because it possesses not only the  unique properties of graphene including  large surface area and flexibility but  also  hydrophilicity and  dispersibility  in aqueous  solutions.  However,there are conflicting results on its biocompatibility and  biosafety partially due to large variations  in  physicochemical  properties  of  GO,  and  the  role  of  these  properties including lateral size in the biological or toxicological effects of GO is still  unclear. In this study, we  focused on the role  of lateral size by  preparing a panel of  GO samples with  differential  lateral  sizes  using  the  same  starting  material.  We  found  that  in comparison to its smaller counterpart, larger GO showed a stronger adsorption onto the plasma membrane with less phagocytosis,  which elicited more robust interaction with toll-like receptors (TLRs) and more potent activation of NF-κB pathways. By contrast,smaller GO sheets were more likely taken up by cells. As a result, larger GO promoted greater  M1   polarization,   associated  with   enhanced  production   of   inflammatory cytokines and  recruitment of  immune cells.  The in vitro  results correlated  well with local  and systemic  inflammatory responses  after  GO administration  into abdominal cavity,  lung  or  blood  stream  through  tail  vein.  Those  findings  are  important  for considering future biological applications of carbon nanomaterials.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/36901
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Recommended Citation:
马娟. 碳纳米材料诱发炎症反应并导致机体损伤的毒性通路研究[D]. 北京. 中国科学院研究生院. 2016.
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