【作者】 陈效；

【导师】 徐顺清； Hermann Schluesener；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生， 2010， 博士

【摘要】 当一种材料的组成颗粒的三维尺寸中至少有一维介于0.1-100纳米之间时,该材料即可称为纳米材料。纳米材料不但具有迥异于宏观材料的理化性能,还可表现出独特的生物学效应；纳米材料的理化特征是决定其生物学效应的关键因素。多壁碳纳米管(MWCNTs)和富勒烯碳60(C60)是碳的两种同素异形体,同时也是具有独特分子结构、理化和生物学性能的纳米材料。MWCNTs和C60与神经组织的相互作用是目前的热点研究领域,并且这两种碳基纳米材料已表现出在中枢神经系统(CNS)的巨大应用潜力。但由于CNS的复杂性,在将MWCNTs和C60安全而有效地引入CNS之前,尚需开展全面和细致的研究以确保其应用的安全性和有效性。星形胶质细胞是CNS十分活跃的细胞成分,其在人脑中的数量十倍于神经元,在神经系统的结构和生理、病理功能方面发挥着至关重要的作用。但目前有关纳米材料与CNS相互作用的研究主要集中于神经元而较少关注胶质细胞。有鉴于此,本课题对原型MWCNTs和C60与大鼠星形胶质细胞和由人星形胶质细胞转化而来的胶质瘤细胞的相互作用开展了研究。本课题包括以下三部分内容。细胞活力检测是任何细胞水平研究的常规和基础性工作。噻唑蓝(MTT)和刃天青(resazurin)还原法是目前广为应用的两种细胞活力检测法。但我们发现,MTT和resazurin法并不能准确反映MWCNTs对细胞活力影响。为能准确检测MWCNTs的细胞效应,我们建立、验证了一系列基于流式细胞技术(FCM)的方法,并行之有效地用于研究MWCNTs对星形胶质细胞的效应。作为对照,C60的细胞效应也经相同的方法进行了分析。JC-1、DiOC6(3)、R123、FDA和PI是反映细胞不同功能的荧光探针。我们首先对它们的染色指征进行了表征。结果表明JC-1和DiOC6 (3)染色是线粒体膜电位(△Ψm)的良好指标。△Ψm是细胞摄取R123的驱动力,但细胞膜转运蛋白Pgp对细胞内R123的含量有决定性的影响。而另一种细胞膜转运蛋白MRP则是细胞FDA染色的决定因素。随后分别以上述探针在原位(in situ)和非原位(ex situ)条件下对经MWCNTs和C60处理(24h)的细胞进行染色,并FCM进行分析。结果显示,在in situ条件下,MWCNTs显著降低JC-1和DiOC6(3)的细胞染色但却增强R123和FDA的染色。而在ex situ条件下,除FDA外,MWCNTs对其他探针的染色无显著影响。与MWCNTs相比,C60对上述探针的细胞染色无显著影响,in situ和ex situ染色结果也无显著差异。显微镜观察显示MWCNTs和C60对细胞形态无明显影响。上述结果表明：1.探针的染色方式对染色结果有着根本影响提示,2.在本实验的暴露浓度和时间的条件下,MWCNTs和C60不影响细胞的总体活力,3.但涉及细胞膜的一些细胞功能可能发生改变,4.荧光探针结合FCM可方便、有效地反映MWCNTs的细胞效应。细胞膜是维持细胞形态和完成细胞生命功能的重要结构,同时也是细胞与纳米材料相互作用的起始界面。跨膜转运以实现细胞内外的物质交换是细胞膜的一个重要功能。细胞膜转运蛋白如Pgp和MRP是完成跨膜物质转运的重要机制。我们在前面的工作中观察到MWCNTs能提高细胞内罗丹明123(R123)和荧光素(Flu)的含量。R123和Flu分别是Pgp和MRP的特异性转运底物,因此推测MWCNTs可能影响细胞膜,尤其是PgP和MRP介导的跨膜转运。在此部分工作中我们即对此假设进行了检验：以MWCNTs处理星形胶质细胞后,分析了细胞对PgP和MRP特异性底物的摄取、潴留和外排动力学。Pgp的底物为R123和阿霉素(DOX); MRP的底物为荧光素(Flu)和羧基荧光素(CF)；并仍以C60作为对照。结果表明,经过MWCNTs处理后,in situ细胞对DOX的摄取显著降低,但对其他药物的摄取无明显改变；同时MWCNTs可显著增加R123、Flu和CF在in situ细胞内的潴留。动力学分析表明MWCNTs明显降低ex situ细胞对R123和Flu的外排速率。与MWCNTs相比,C60对各底物的细胞摄取、潴留以及外排速率无明显影响。上述结果提示MWCNTs可影响跨细胞膜物质转运,特别是Pgp和MRP介导的跨膜转运。前面的工作利用大鼠星形胶质细胞模型表明MWCNTs可抑制细胞膜PgP和MRP介导的跨膜转运。PgP和MRP在人体内有广泛的分布并有重要的生理和病理意义。尤其值得重视的是,肿瘤细胞过度表达PgP和MRP是肿瘤多药耐药(MDR)现象的重要机制。因此在人肿瘤细胞中研究MWCNTs对Pgp和MRP的影响更具实际意义和价值。鉴于此,此部分工作在MDR人脑胶质瘤细胞中验证了MWCNTs对Pgp和MRP的效应,并围绕MRP对MWCNTs的效应进行了初步的机制研究。MRP的转运功能依赖于还原型谷胱甘肽(GSH)的参与。同时,细胞膜电位也是Flu外排的重要调控因素。验证实验表明,减少细胞内的GSH和改变细胞膜电位均可影响胶质瘤细胞对Flu的外排转运。我们在经MWCNTs处理的胶质瘤细胞中检测到大量活性氧(ROS),同时细胞内的GSH水平显著降低。两种细胞膜电位探针(DiBAC4(3)和DiOC6(3))染色分析也显示MWCNT可降低细胞膜电位。上述结果提示MWCNTs对MRP的影响机制涉及细胞膜电位改变和氧化应激所致的GSH减少。总结1.研究方法和手段的选择、验证和优化对于纳米材料的生物学效应研究尤为重要。本课题表明流式细胞技术是适用于MWCNTs细胞效应研究的良好手段。但在应用中仍应全面考虑材料本身、细胞模型和研究方法等方面存在的影响因素,以获得准确的结论。2.纳米材料的形态结构对其生物活性有着决定性的影响。本课题表明碳基纳米材料MWCNTs可影响跨细胞膜物质转运,尤其是抑制PgP和MRP介导的跨膜转运。而同为大分子碳,但纳米结构不同的C60却无此效应。3. MWCNTs对MRP的影响机制可能涉及细胞内GSH耗竭和细胞膜电位改变。更多还原

【Abstract】 Nanomaterials refer to materials that have at least one dimension 100 nanometres or less. Materials at nanoscale not only possess extraordinary physic-chemical properties not seen at macroscale but also can have unique biological effects which are foundamentally determined by their physic-chemistry. A best pair of examples is multiwalled carbon nanotubes (MWCNTs) and flullerene C60 (C60) which are carbon allotropes of distinct nano structures. Neuroscience is a field where the interactions of theses two types of carbon macromolecules with neverous system are a focus of research attention and where the biological properties and application potential of MCNTs and C60 are most extensively explored. Astrocytes, or astroglial cells, are a dominant and highly active component of the central nervous system (CNS) that play pivotal structural and functional roles and outnumber neurons by about 10 fold in the human brain. The purpose of the present project was to investigate the interactions of MWCNTs and C60 with astrocytes and transformed astrocytes i.e. glioma cells and to compare the dfferent biological effects of MWCNTs and C60.Monitoring of cell viability is a regular but essential part of work in any cell-based study. Two widely used assays i.e. MTT reduction and resazurin reduction were first performed to evaluate the effects of MWCNTs and C60 on the viability of in vitro rat astrocytes, but yielded contradictory results regarding MWCNTs, indiating unsuitability of these methods for assaying cellular responses to MWCNTs. To provide accurate information on MWCNTs’ cellular effects, a series of flow cytometry-based procedures were then developed, validated and effectively applied. C60 was also tested in comparison with MWCNTs. Briefly, cells exposed to MWCNTs were stained by a panel of fluorescent probes for different aspects of cell function, including 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide (JC-1),3,3’-Dihexyloxacarbocyanine, iodide (DiOC6(3)), rhodamine 123 (R123) and fluorescein diacetate (FDA). Probe indications were characterized and probe staining performed both in an "in situ" and "ex situ" manner on MWCNTs-or C60-exposed cells were analyzed by flow cytometry. JC-1 and DiOC6 (3) staining were found good indicators of mitochondrial membrane potential (ATm). WhileΔ(?)m contributed to R123 uptake, intracellular accumulation of R123 was largely determined by the function of P-glycoprotein, a cell membrane bound efflux protein. Multidrug-resistance associated protein, another efflux protein, was found to determine cellular FDA staining. Decreased JC-1 and DiOC6(3) uptake were observed in MWCNTs-exposed cells but couldn’t be attributed toΔ(?)m disruption. In contrast, cellular staining of R123 and FDA was enhanced after MWCNT exposure. Mode of dye loading was found to significantly affect the outcome of cellular dye staining after MWCNT exposure. Compared with MWCNTs, C60 generally exerted insignificant influence on the staining of all probes. In summary, fluorescent probe staining in combination with flow cytometry, after careful validation, can provide good assay of cellular reposnses to the exposure of MWCNTs and C60. Astrocyte viability appeared to be maintained in the presence of MWCNTs and C60 at concentrations and for the duration tested. However, certain aspect of cell function relating to the cell membrane might be affected.One of the many functions of the cell membrane is to execute substance transport into and out of the cell and cell membrane transporter proteins like P-glycoprotein (Pgp) and multidrug resistance related protein (MRP) are a key mechanism therefor. As observed in my work presented in Part 1, rat astrocytes exposed to MWCNTs but not C60 displayed increased contents of rhodamine 123 (R123) and fluorescein (Flu) which are respective substrates of Pgp and MRP. It was thus postulated that MWCNTs might be able to affect cross-membrane drug transport, particularly that mediated by Pgp and MRP. To test this hypothesis, we investigated the influence of MWCNTs, and C60 as a comparison, on the transport of several compounds across the cell membrane of rat astrocytes using flow cytometry. These compounds were fluorescein diacetate, carboxyfluorescein diacetate, rhodamine 123 and doxorubicin which are prosubstrate/substrates of multidrug transporter proteins. Results showed that MWCNTs significantly inhibited cellular uptake of doxorubicin but not the other drugs and mode of loading made a significant difference in doxorubicin uptake. Retention of fluorescein, carboxyfluorescein and rhodamine 123 was remarkably higher in MWCNTs-exposed cells after an efflux period. Kinetics study also demonstrated slowed efflux of intracellular fluorescein and rhodamine 123. C60 generally had no siginificant effect on the uptake and efflux of the tested substrates. Data presented in this paper suggest that MWCNTs could affect drug transport across cell membrane.MRP-mediated drug transport in a human glioma cell model In the study on the interactions of carbon nanotubes with living cells, the cell membrane deserves particular attention as it provides the first interface to initiate CNTs-cell interactions. In my work presented in Part 2, MWCNTs were demonstrated to be able to affect cross-membrane drug transport, especially that mediated by Pgp and MRP in a rat astrocyte model. In the present work, the inhibiting effect of MWCNTs on the MRP mediated fluorescein efflux in a human glioma cell model was also demonstrated. To provide clues to explanation of this effect, intracellular glutathione content and reactive oxygen species production were determined as fluorescein is a specific substrate of cell membrane MRP whose transport activity requires glutathione which can be depleted under oxidative stress. The plasma membrane potential was also probed as the susceptibility of fluorescein efflux to modulation of the plasma membrane potential has been documented. Results showed a remarkable decrease in cellular glutathione level as well as an increase in reactive oxygen species production. Probe staining also indicated decreased plasma membrane potential. The data suggested that multiwalled carbon nanotubes may affect the transport activity of cell membrane multidrug resistance-related protein through reduction of intracellular glutathione content. Hypopolarization of the plasma membrane may also contribute to MWCNTs’effect. In vitro rat astrocytes and human glioma cells were studied for their interactions with prestine MWCNTs and C60. Principle and original discoveries of my work are 1) Investigation methods and approaches commonly used for conventional drug studies might yield misleading results when used for biological study of nanomaterials like MWCNTs. Optimized and validated flow cytometry based assays are convenient and effective for studying cellular effects of nanomaterials like MWCNTs. Yet, caution and discretion are still needed to ward off false judgements.2) Prestine MWCNTs and C60 both have little influence on cell viability, but drug transport across cell membrane, particularly that mediated by transporter proteins can be affected by MWCNTs but not C60. MWCNTs and C60 are both carbon macromolecules. But they can have distinct biological effects as demonstrated in the present work, probably due to their different nanostructures.3) The mechanisms of MWCNTs’effects may involve cell membrane perturbation and depletion of intracellular glutathione due to oxidatice stress.更多还原