碳纳米管致血管内皮细胞损伤的作用及其分子调控机制
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摘要
研究目的:
随着纳米材料的日益广泛应用,纳米材料已逐步大量进入工作和生活环境,纳米产品生产者和消费者的暴露机会逐步增多。这意味着纳米材料和纳米颗粒将有更多的机会与血管、血液及其中的成分发生相互作用,从而对人类健康造成影响。然而由于目前相关研究证据极少,尚不能明确纳米材料物理化学参数(形状、大小、尺寸分布、表面结构、电化学特性等)与心血管系统毒性作用的关系,心血管毒性产生的直接或间接作用及其分子调控机制。由于心血管疾病属于人类重大疾病,严重威胁人类健康与生命,因而开展典型纳米材料暴露所致心血管毒作用及其机制的研究,可以更好地利用纳米材料的正面效应,为预防、减少或消除其对健康可能产生的不良影响,为纳米材料安全性评价技术与标准的建立提供理论和技术基础。本课题选择碳纳米管作为研究纳米材料致心血管系统毒性作用的典型目标分子。
依据纳米材料可以进入血液循环以及大气颗粒物流行病学研究结果,可以推断并提出假设,处于纳米尺度的人造纳米材料也可产生心血管毒性作用并且其毒性作用可能更为严重。基于对现有文献资料的分析以及我们前期工作的积累,结合了解纳米材料的特殊性质和应用情况,提出本项目“碳纳米管致血管内皮细胞损伤的作用及其分子调控机制”,探讨纳米材料对心血管系统毒作用及其分子调控机制。选择血管内皮细胞作为靶细胞,原因之一是从主动脉平面结构分析,血管内皮细胞充当阻止血液耐受的毒素或化合物从血管管腔进入血管壁深层的第一道细胞屏障,因而也将成为进入血液的纳米颗粒进攻的靶点。另外,研究证实内皮细胞的机械性损伤或毒性损伤与动脉粥样硬化反应的启动有关,一旦血管内皮细胞受损,一方面会增加其通透性而失去屏障功能,另一方面则会使促栓物质生成较多及抗凝物质生成减少,空泡形成、血小板减少,导致各种血栓性疾病的发生。
动脉粥样硬化是一种慢性炎症增殖性疾病,血管内皮细胞的慢性损伤是动脉粥样硬化发生的先决条件及重要机制。单核细胞穿过动脉内皮层进入内膜是动脉粥样硬化病变形成早期事件之一,这一过程涉及单个核细胞-血管内皮细胞表面诸多分子之间的相互作用,其中血管内皮细胞分泌的单核细胞趋化蛋白-1(monocyte chemoattractant protein-1)等趋化因子和血管细胞粘附因子-1(vascular cell adhesive molecule-1)、细胞间粘附因子-1(intercellular adhesive molecule-1)等粘附分子在介导单个核细胞向血管内皮细胞募集、粘附、迁移过程中起了极其重要的作用。血管内皮细胞功能受损过程中分泌了大量的细胞因子,它们通过细胞间的网式相互作用而不是单独起作用,一个因子的释放可导致第二个因子的表达,不仅作用于临近细胞,而且还可以再作用于自身。核因子-kB(nuclear factor-kB, NF-kB)通过调控血管内皮细胞中各种炎性和免疫因子的表达来调控各种细胞因子的表达。我们采用western blot方法和免疫组化法研究碳纳米管对NF-kB相关蛋白表达的影响,探讨碳纳米管致血管内皮细胞损伤作用可能参与的信号通路,阐明其损伤的分子机理。
本研究的目的在于认识碳纳米管致血管内皮细胞损伤的生物学规律,揭示碳纳米管损伤血管内皮细胞的生物信号转导途径和关键介导因子,以阐明碳纳米管血管损伤的生物学基础;认识碳纳米管诱导血管内皮细胞粘附因子ICAM-1和VCAM-1水平的变化及其与心血管疾病发生的关系,以阐明在碳纳米管作用下ICAM-1、VCAM-1水平变化的机制及其分子基础,为碳纳米管潜在的生物安全性评价提供实验依据,为探索有效的预防措施提供基础。
研究方法:
利用整体动物实验和细胞实验相结合的方法,从整体水平和细胞水平探讨碳纳米管对机体的毒性作用。相关指标包括氧化应激、炎症因子、粘附分子的测定。利用生化分析、免疫学方法、RT- PCR技术以及免疫印迹方法从蛋白和基因的水平对实验数据进行综合分析。
研究结果:
1整体动物水平实验:选取Wistar雄性大鼠,体重180±20g,设置3.5mg/kg和17.5mg/kg两个剂量组,以生理盐水作空白对照,同时采用纳米级的炭黑和石英分别做阴性对照组和阳性对照组。本实验共设7个组(空白对照组,炭黑高、低剂量组,碳纳米管高、低剂量组,SiO2高、低剂量组),每组10只大鼠,分别于连续染毒7d和30d后处死,取相关组织进行检测。
1)病理损伤检测结果:动物处死后取肺、肝、肾和主动脉作病理学检查。检测结果表明,除肾没有病理变化外,其余各器官均有病理损伤。在相同的染毒剂量条件下,30d病理损伤程度较7d严重,而且碳纳米管组的损伤程度较其它各组严重;在相同的染毒时间条件下,高剂量组的病理损伤程度较低剂量组严重,而且也是碳纳米管的损伤最严重。主动脉病理检测结果表明,只有碳纳米管30d处理组有动脉壁突起现象。
2)血常规和血生化检测结果:血常规检测指标中只有白细胞(WBC)计数、血小板(PLT)计数和全血黏度值(ηb)水平有变化,而且在各暴露组中具有时间效应和剂量效应关系;血生化检测指标中LDH活性,CK、AST、ALT和sEPCR含量五项指标均有变化,在各暴露组中也同样存在时间效应和剂量效应关系。
3)氧化应激检测结果:检测血清中GSH和O2ˉ·水平,碳纳米管高剂量组GSH水平下降明显较其它处理组高,SiO2高剂量处理组O2ˉ·水平最高。总之,碳纳米管暴露组在相同的剂量和染毒时间条件下明显较空白对照组和阴性对照组高。
4)血清中细胞因子TNF-α和IL-1β检测结果:随着染毒剂量的增加和染毒时间的延长,血清中TNF-α和IL-1β水平呈现一定的剂量效应和时间效应关系。而且在碳纳米管高剂量暴露组含量最高。
5)血清中细胞粘附分子ICAM-1和VCAM-1检测结果:随着染毒剂量的增加和染毒时间的延长,血清中ICAM-1和VCAM-1水平呈现一定的剂量效应和时间效应关系。而且碳纳米管高剂量暴露组水平最高。
6)免疫组化实验结果:利用免疫组化方法对主动脉壁的突起部分进行ICAM-1和VCAM-1表达的检测,结果表明高剂量碳纳米管30d处理组主动脉壁上ICAM-1的表达强于VCAM-1 ,但二者均明显高于空白对照组。
2.细胞水平实验:将碳纳米管配制成由高到低依次为200、100、50、25、12.5、6.25、3.12μg/ml的悬液,分别染毒传代培养2-3代的大鼠主动脉血管内皮细胞,利用细胞培养液作为空白对照,测试指标与结果如下:
1)氧化应激:分别于染毒结束后检测细胞培养液上清中LDH活性、GSH水平。细胞内LDH活性在碳纳米管染毒浓度为0~100μg·mL-1范围内随染毒剂量的增加而逐渐上升,在200μg·mL-1剂量组,其释放有所减弱;而培养液上清中GSH在碳纳米管染毒浓度为0~200μg·mL-1范围内,其含量随染毒剂量的增加而逐渐下降。
2)细胞超微结构的改变:电镜扫描结果表明,碳纳米管能够穿透血管内皮细胞膜进入细胞质并在其中聚集,进而对细胞器产生损伤作用。在100μg·mL-1剂量暴露12h组表现内质网及线粒体略肿胀,细胞溶酶体增加,染色质边染,但细胞膜完整;而在暴露20h组内皮细胞形态不完整,大部分RAECs水肿,坏死、脱落基底膜裸露,有些区域甚至断裂,核皱缩;线粒体明显肿胀,形状不规则空泡变,外膜模糊,内嵴不清,基底致密等退行性变;小部分区域内内皮细胞较完整,病变轻,仅细胞内线粒体略肿胀,胞浆基质疏松变淡,线粒体空泡变性,粗面内质网扩张,其内脂滴增多,内皮细胞间连接处微绒毛增多。
3)细胞存活率的改变:利用MTT实验方法检测不同浓度碳纳米管暴露不同时间后,大鼠主动脉血管内皮细胞存活率的改变。结果表明,随着碳纳米管染毒浓度的上升和染毒时间的延长,大鼠主动脉血管内皮细胞存活率逐渐下降,死亡率逐渐上升。
4)单个核细胞-血管内皮细胞粘附:电镜下RAECs呈单层、紧密贴壁,呈爬行式生长。细胞边缘不整齐,表面不光滑,有丝状或薄翼状的突起。PBMCs呈圆球形,表面不光滑,呈绒毛状。电镜下可见RAECs通过表面的突起粘附PBMCs,PBMCs也通过表面的绒毛与RAECs粘附。观察到“间接系链”(secondary-tethering)现象,即粘附在RAECs表面的PBMCs之间也有相互连接,或者是粘附在RAECs表面的PBMCs作为支撑点粘附了其它的PBMCs,结果使得RAECs与PBMCs的粘附加强和扩大;而且随着染毒剂量的增加大鼠主动脉血管内皮细胞与单个核细胞的粘附率显著增加。
5)细胞粘附分子ICAM-1和VCAM-1蛋白及mRNA的表达测定:利用ELISA试剂盒检测不同时间和不同浓度碳纳米管暴露后细胞培养液上清中细胞粘附分子ICAM-1和VCAM-1蛋白的表达;利用RT-PCR技术测定不同时间和不同浓度碳纳米管暴露后细胞ICAM-1和VCAM-1mRNA的表达。结果表明随着碳纳米管暴露时间和暴露浓度的增加,ICAM-1和VCAM-1mRNA和蛋白的表达均呈增加趋势,具有一定的时间效应和剂量效应关系。
6) IκKα、IκBα检测:为了进一步验证碳纳米管暴露致大鼠主动脉血管内皮细胞损伤的NF-kB途径,我们利用免疫印迹技术对IKKα、IκBα及NF-κB的表达进行检测,结果表明随着碳纳米管暴露剂量的增加,IKKα及NF-κB的表达呈上升趋势,而IκBα的表达呈下降趋势。
7)NF-kB/P65的核移位表达:利用免疫细胞化学方法对核转录因子NF-kB/P65的核移位表达进行检测,碳纳米管暴露组明显高于空白对照组和N-乙酰水杨酸预处理组,说明碳纳米管是通过致细胞炎症反应而致细胞损伤,而且是通过激活NF-kB/P65途径进行的。
研究结论:
1.整体动物实验结果表明碳纳米管暴露可致大鼠主动脉血管壁损伤,机体出现明显的氧化应激和炎症反应,血中前炎症因子TNF-α和IL-1β及反映血管内皮细胞功能的可溶性细胞间粘附因子(sICAM-1)和可溶性血管细胞粘附因子(sVCAM-1)较空白对照组和阴性对照组均明显升高。
2.细胞水平实验结果表明,碳纳米管暴露可致大鼠主动脉血管内皮细胞损伤。表现为细胞超微结构的改变和细胞存活率的下降,特别是作为血管内皮细胞功能紊乱的评价指标ICAM-1和VCAM-1 ,碳纳米管暴露后其mRNA和蛋白均有过度的表达,利用免疫印迹技术和免疫组化技术检测与这两种粘附分子表达相关的核转录因子NF-κB相关蛋白也呈现过度表达。
3.激活NF-κB信号通路途径:细胞水平实验表明活性氧激活血管内皮细胞内的核转录因子κB,启动血管内皮细胞内核转录因子κB信号途径而增加血管内皮细胞表面细胞间粘附分子-1的表达,介导中性粒细胞与血管内皮细胞的牢固粘附;而整体动物实验则是通过CNTs暴露致机体产生活性氧从而激活细胞内核转录因子κB信号途径,同时活性氧释放到细胞外,刺激巨噬细胞和T淋巴细胞分泌TNFα和IL-1β,在TNFα、IL-1β与核转录因子κB的协同作用下,血管内皮细胞中粘附分子表达增加,诱导并加强中性粒细胞与血管内皮细胞的粘附。
The most attractive properties of nanomaterials for medical and technologic applications, including their small size, large surface area, and high reactivity, are also the main factors for their potential toxicity. Based on their inhalation studies with ambient ultrafine particles, it has been predicted that nanosized particles will have higher pulmonary deposition and biological activity compared with larger particles. Thus, some nanosized materials may induce not only damage at the deposition site but also distant responses as a result of their translocation and/or reactivity through the body. In this respect, epidemiologic and experimental studies have suggested an relation between respiratory exposure to ambient ultrafine particles ( including particles with a diameter <100nm ) and the progression of cardiovascular disease. Engineered carbon nanomaterials, including carbon nanotubes, have elicited a great deal of interest recently because of their unique electronic and mechnical properties. Carbon nanotubes(CNT ) have a diameter ranging from 0.7 to 1.5nm, with lengths >1μm. Initial toxicologic studies demonstrated that intratracheal or pharyngeal instillation of CNTs suspension in mice caused a persistent accumulation of carbon nanotube aggregates in the lung, followed by the rapid formation of pulmonary granulomatous and fibrobic tissues at the site. The unique physical characteristic and the pulmonary toxicity of CNTs raised concerns that respiratory exposure to these materials might be associated with systemic toxicities. In the present studies, we demonstrate that lung instillation of CNTs is associated with a dose-dependent increase in oxidative modifications, which are considered to play a role in atherogenesis, we further evaluated the effects of CNTs respiratory exposure on rat.
Since endothelium is the first layer of vascular, endothelial cells would be the target to be injured firstly. The cell adhesion molecules including ICAM-1 and VCAM-1 are important for binding of leukocytes to the endothelial cells and in the infiltration of inflammatory cells into tissues. Nuclear factor-kB (NF-kB) has been implicated in the transcriptional activation of the genes encoding CAMs. Rapid phosphorylation and degradation of IkBαallows NF-κB to translocate into the nucleus and to regulate transcription of the target genes. As very little is known in regard to the toxicology and the underlying mechanism involved in explaining the phenomena of carbon nanotubes (CNTs) exposure, we carried out a detailed study on their effects of the expression of cell adhesion molecules on rat aortic endothelial cells ( RAECs). We demonstrate here that CNTs induced adhesion of neutrophils to endothelial monolayer by promoting the expression of ICAM-1, VCAM-1. Since NF-κB is a major transcription factor involved in the transcriptional regulation of cell adhesion molecules, thus we studied the status of NF-κB/P65 activation in CNTs treated RAECs. As oxidative stress is known to regulate the activation of NF-κB, we tested GSH content and LDH activity. We found that CNTs induced oxidative stress and调the expression of ICAM-1 and VCAM-1. The nucleus translocation of NF-κB/P65 after CNTs exposure could be inhibited by N-acetylcysteine which indicated that the high expression of ICAM-1 and VCAM-1 mediated by oxidative stress in rat aortic endothelial cells might play an important inflammation role in CNTs-induced vascular endothelium damage. After establishing RAECs damage model, we measured the expression of ICAM-1 and VCAM-1 in cultured RAECs stimulated with different dose of CNTs for different time exposure and detected NF-κB signaling pathways.
Method :
we evaluated the toxic effects of intratracheal instillation of CNTs on rats and CNTs exposure on RAECs. The related indexes including oxidative damage, inflammatory factor, adhesive molecular and signal molecular were detected by biochemical assays, immunological histochemistry, RT-PCR and Western blot. Data analyzed by using SPSS1 .0 software.
Result:
1. Animal test:
Rats were intratracheally instilled with 0, 0.7, or 3.5 mg of carbon nanotubes, carbon black as negative control, or quartz as positive control. The rats were euthanized on 7 d or 30 d after the single treatment per day for study of histopathological changes in some organs and expression of sICAM-1 and sVCAM-1in rats’blood.
1)Histopathological examination:
Histopathological evaluation of lung tissues revealed that lung in rats exposed to CNTs and quartz particles produced a dose-dependent lung inflammatory response characterized by neutrophils infiltration and foamy (lipid-containing) alveolar macrophage accumulation. In addition, lung tissue thickening as a prelude to the development of fibrosis was evident and progressive. Histopathological evaluation of aortic vascular tissue revealed that CNTs exposure fior 30d resulted in a protuberance on inner membrane of aortic vascular, but CB and SiO2 groups had no this kind phenomenon.
2)Blood routine examination and blood biochemistry assay:
The results of blood routine examination showed that only the contents of PLT、WBC and ?b value were different between NS control group and CNTs、SiO2 experimental groups, especially in CNTs experimental group these index showed the highest value. The results of LDH activity, CK、ALT、AST and sEPCR contents increased in a dose dependant manner and time dependant manner under treatment with all particle types especially the highest release induced by SiO2 at a particle concentration of 17.5 mg/kg. However, maximal LDH release induced by CNTs treatments was noted to be lower than that observed with SiO2 (positive control) possibly due to high particle concentrations interfering with the assay.
3)Oxidative stress and formation of reactive oxygen species:
A significant depletion of GSH was observed at high dose of CNT exposure compared with control group, and was also time- dependent. But the content of O2ˉ·increased with the time and dose of CB、CNTs and SiO2 exposure, and a significant increase in CNTs and SiO2 exposure compared with control group. Overall, the results demonstrated a significant depletion of GSH and increase in oxidative damage levels occurred in CNTs exposed rats.
4)Inflammatory response (TNF-αand IL-1β):
Our results showed that CNTs exposure elevated the expression of TNF-αand IL-βin dose and time dependent manner, although there was only weak expression in low dose group.And in the case of the same dose treatment, the longer the CNTs exposure, the higher expression level of them occurred, and the expression level of these two inflammatory factors in rats exposed to CNTs exposure was greater than that of CB and SiO2 at the same dose and the same time.
5)sICAM-1 and sVCAM-1 expression of blood:
CNTs increased monocyte adhesion to cells dose-dependently and time-dependently. Meanwhile, CNTs exposure elevated the expression of sICAM-1 and sVCAM-1 in dose and time dependent manner, although there was only weak expression in low dose group.And in the case of the same dose treatment, the longer the CNTs exposure, the higher expression level of them occurred, and the expression level of these two adhesion molecular in rats exposed to CNTs was greater than that of CB and SiO2 at the same dose and the same time.
6)Immunohistochemistry of ICAM-1 and VCAM-1 on aortic vascular after CNT
exposure:
The results of immunohistochemistry showed that ICAM-1 and VCAM-1 only expressed infirmly in endothelial cells of NS control group , but VCAM-1 expressed positively in endothelial cells and protuberance of CNTs experiment group, which were located in cytoplasm, cytomembrane and intercells; ICAM-1 expressed firmly in protuberance including endothelial cells even smooth muscle cells of midmembrane.
2.Cell test:
RAECs were exposed to CNTs suspension at different dose and for different time.
1)In vitro cell oxidative stress: A significant depletion of GSH was observed at dose 50-200μg·mL-1 of CNTs compared with that in the control group. Overall, the results demonstrated a significant depletion of GSH levels occurred in RAECs exposed to CNTs. The results of LDH leakage and exhibited a significant cytotoxicity at CNTs concentrations of 12.5-100μg·mL-1. There was a statistically significant difference between different dose of CNTs, whereas the highest toxicity was at 100μg·mL-1 but not 200μg·mL-1.The detailed mechanism is not clear, which needs more work to do.
2)CNTs localization and ultrastructure observation: After exposure to CNTs, there were clear changes in the ultramicroscopic features of RAECs. CNTs frequently lodged inside cytoplasm and mitochondria. RAECs incubated with CNTs showed extensive disruption of mitochondrial cristae, resulting in a vacuolar cellular appearance and microvillus. These changes were time dependent, with fewer particles collecting inside mitochondria during shorter incubations. Electron microscopy showed considerable mitochondrial damage by CNTs, resulting in the formation of concentric structures, known as myelin figures.
3)Cell viability:
The MTT assay showed CNTs was toxic to RAECs and the cytotoxicity increased with CNTs doses and exposure time. From these results, we could see that most mitochondria were damaged characteristically by swelling with architectural disruption which was consistent with the qualitative analysis.
4)SEM image of adhesion between RAEC and PMBC after CNTs exposure:
Under the SEM, RAECs grew as a monolayer, adhering to culture bottle wall like creeping. Cells’border was not tidy, surface not smooth and had silk-like salience. RAECs could connect each other by these saliences. PBMC were like ball and their surfaces were not smooth which looked like microvillus. RAECs adhered to PMBCs through these saliences under SEM. PMBCs could also adhere to RAECs by microvillus on their surfaces. Secondary-tethering phenomenon could be observed: connection between PMBCs which jointed to the surfaces of RAECs or backstop adhered to the surface of RAECs provided a point to adhere to other PBMCs which led to strengthen and enlarge adhesion between RAECs and PMBCs. The degree of adhesion increased with the doses of CNTs.
5)Expression of ICAM-1 and VCAM-1 protein and mRNA:
CNTs exposure elevated the expressions of ICAM-1 and VCAM-1 in dose and time dependent manner.By one way analysis of variance,the expression level of ICAM-1 and VCAM-1 in vascular cellular endothelium in high dose groups (25-200ug·mL-1) was statistical higher than that in the control group.And in the case of the same dose treatment, the longer the CNTs exposure, the higher expression level of them occurred, but the difference of expression level between exposure for 12h and 24h was greater than that for 24h and 48h. In agreement with the protein data, noticeable changes were detected in the steady-state mRNA levels of both ICAM-1 and VCAM-1.
6) CNTs exposure induced activation of NF-κB, IKKαand the degradation of IκBα:
Comparison of the levels of NF-κB in the nuclear fraction indicated that the level of NF-κB in the nuclear fraction was higher in CNTs exposure cells than that in the control cells. Western blotting and densitometric analysis of the density of bands relative toβ-actin indicated that CNTs exposure resulted in markedly greater activation of NF-κB and its translocation to the nucleus than that in control RAECs. However, the CNTs induced activation and translocation of NF-κB to the nucleus in the RAECs was greater than that in the control RAECs; Western blot analysis and subsequent measurement of density of bands relative toβ-actin indicated that activation level of IKKαwas higher in CNTs exposure RAECs than that in the control RAECs. Greater degradation of IκBαwas noted in CNTs exposure RAECs as compared to the control RAECs.
7)Effects of N- acetylcystein on nuclear translocated NF-κB/p65 induced by CNTs in RAECs:
The immunoanalysis of effects of arecholine on nuclear translocated NF-κB/p65 induced by CNTs in RAECs showed that CNTs exposure increased the nuclear translocated NF-κB/p65 compared with that in the control group, however pretreating the cells with N-acetylcystein before CNTs exposure, the nuclear translocated NF-κB/p65 decreased but was still higher than that in the control group. It suggested that N-acetylcysteine could inhibit CNTs to induce NF-κB/p65 expression on rat aortic endothelial cells. ROS production and oxidative stress were probably some of the mechanisms.
Conclusion:
1. An important consideration is that oxidative stress, and especially depletion of reduced GSH and O2ˉ·production, the leakage of IL-1βand TNF-αincreasing with dose and time of CNTs exposure, which is the same as the results of sICAM-1 and sVCAM-1. These results show that, for the test conditions described here and on an equal-weight basis, carbon nanotubes can pass through lung-blood barrier and reach the blood, they are much more toxic than carbon black and can be more toxic than quartz, which is considered a serious occupational health hazard in chronic inhalation exposures. As markers of endothelial cells damage, the three indicators of experimental animals especially CNT group are higher than those of control group. In this study, we have sought to bring these findings to suggest pathobiologic processes whereby nanaoparticles, especially CNTs, had effects on the cardiovascular system. Pathologic end points relevant to plaque rupture, endothelial erosion, hemostasis, and coagulation should be used in toxicologic studies.
2. The results of cell tests showed that CNTs exposure can induce RAECs damage which including ultrastructure of RAECs changed and cell viability decreased, especially the expression of ICAM-1 and VCAM-1 mRNA and protein are higher than that in control RAECs.
3. We demonstrate here that CNTs induce adhesion of neutrophils to endothelial monolayer by promoting the expression of ICAM-1, VCAM-1. Since NF-κB is a major transcription factor involved in the transcriptional regulation of cell adhesion molecules, thus we studied the status of NF-κB/P65 activation in CNTs treated RAECs. As oxidative stress is known to regulate the activation of NF-κB, we tested GSH content and LDH activity. We found that CNTs induced oxidative stress and the expression of ICAM-1 and VCAM-1. The nucleus translocation of NF-κB/P65 after CNTs exposure can be inhibited by N-acetylcysteine, which indicate that the high expression of ICAM-1 and VCAM-1 mediated by oxidative stress in rat aortic endothelial cells may play an important inflammation role in CNT-induced vascular endothelium damage.
随着纳米材料的日益广泛应用,纳米材料已逐步大量进入工作和生活环境,纳米产品生产者和消费者的暴露机会逐步增多。这意味着纳米材料和纳米颗粒将有更多的机会与血管、血液及其中的成分发生相互作用,从而对人类健康造成影响。然而由于目前相关研究证据极少,尚不能明确纳米材料物理化学参数(形状、大小、尺寸分布、表面结构、电化学特性等)与心血管系统毒性作用的关系,心血管毒性产生的直接或间接作用及其分子调控机制。由于心血管疾病属于人类重大疾病,严重威胁人类健康与生命,因而开展典型纳米材料暴露所致心血管毒作用及其机制的研究,可以更好地利用纳米材料的正面效应,为预防、减少或消除其对健康可能产生的不良影响,为纳米材料安全性评价技术与标准的建立提供理论和技术基础。本课题选择碳纳米管作为研究纳米材料致心血管系统毒性作用的典型目标分子。
依据纳米材料可以进入血液循环以及大气颗粒物流行病学研究结果,可以推断并提出假设,处于纳米尺度的人造纳米材料也可产生心血管毒性作用并且其毒性作用可能更为严重。基于对现有文献资料的分析以及我们前期工作的积累,结合了解纳米材料的特殊性质和应用情况,提出本项目“碳纳米管致血管内皮细胞损伤的作用及其分子调控机制”,探讨纳米材料对心血管系统毒作用及其分子调控机制。选择血管内皮细胞作为靶细胞,原因之一是从主动脉平面结构分析,血管内皮细胞充当阻止血液耐受的毒素或化合物从血管管腔进入血管壁深层的第一道细胞屏障,因而也将成为进入血液的纳米颗粒进攻的靶点。另外,研究证实内皮细胞的机械性损伤或毒性损伤与动脉粥样硬化反应的启动有关,一旦血管内皮细胞受损,一方面会增加其通透性而失去屏障功能,另一方面则会使促栓物质生成较多及抗凝物质生成减少,空泡形成、血小板减少,导致各种血栓性疾病的发生。
动脉粥样硬化是一种慢性炎症增殖性疾病,血管内皮细胞的慢性损伤是动脉粥样硬化发生的先决条件及重要机制。单核细胞穿过动脉内皮层进入内膜是动脉粥样硬化病变形成早期事件之一,这一过程涉及单个核细胞-血管内皮细胞表面诸多分子之间的相互作用,其中血管内皮细胞分泌的单核细胞趋化蛋白-1(monocyte chemoattractant protein-1)等趋化因子和血管细胞粘附因子-1(vascular cell adhesive molecule-1)、细胞间粘附因子-1(intercellular adhesive molecule-1)等粘附分子在介导单个核细胞向血管内皮细胞募集、粘附、迁移过程中起了极其重要的作用。血管内皮细胞功能受损过程中分泌了大量的细胞因子,它们通过细胞间的网式相互作用而不是单独起作用,一个因子的释放可导致第二个因子的表达,不仅作用于临近细胞,而且还可以再作用于自身。核因子-kB(nuclear factor-kB, NF-kB)通过调控血管内皮细胞中各种炎性和免疫因子的表达来调控各种细胞因子的表达。我们采用western blot方法和免疫组化法研究碳纳米管对NF-kB相关蛋白表达的影响,探讨碳纳米管致血管内皮细胞损伤作用可能参与的信号通路,阐明其损伤的分子机理。
本研究的目的在于认识碳纳米管致血管内皮细胞损伤的生物学规律,揭示碳纳米管损伤血管内皮细胞的生物信号转导途径和关键介导因子,以阐明碳纳米管血管损伤的生物学基础;认识碳纳米管诱导血管内皮细胞粘附因子ICAM-1和VCAM-1水平的变化及其与心血管疾病发生的关系,以阐明在碳纳米管作用下ICAM-1、VCAM-1水平变化的机制及其分子基础,为碳纳米管潜在的生物安全性评价提供实验依据,为探索有效的预防措施提供基础。
研究方法:
利用整体动物实验和细胞实验相结合的方法,从整体水平和细胞水平探讨碳纳米管对机体的毒性作用。相关指标包括氧化应激、炎症因子、粘附分子的测定。利用生化分析、免疫学方法、RT- PCR技术以及免疫印迹方法从蛋白和基因的水平对实验数据进行综合分析。
研究结果:
1整体动物水平实验:选取Wistar雄性大鼠,体重180±20g,设置3.5mg/kg和17.5mg/kg两个剂量组,以生理盐水作空白对照,同时采用纳米级的炭黑和石英分别做阴性对照组和阳性对照组。本实验共设7个组(空白对照组,炭黑高、低剂量组,碳纳米管高、低剂量组,SiO2高、低剂量组),每组10只大鼠,分别于连续染毒7d和30d后处死,取相关组织进行检测。
1)病理损伤检测结果:动物处死后取肺、肝、肾和主动脉作病理学检查。检测结果表明,除肾没有病理变化外,其余各器官均有病理损伤。在相同的染毒剂量条件下,30d病理损伤程度较7d严重,而且碳纳米管组的损伤程度较其它各组严重;在相同的染毒时间条件下,高剂量组的病理损伤程度较低剂量组严重,而且也是碳纳米管的损伤最严重。主动脉病理检测结果表明,只有碳纳米管30d处理组有动脉壁突起现象。
2)血常规和血生化检测结果:血常规检测指标中只有白细胞(WBC)计数、血小板(PLT)计数和全血黏度值(ηb)水平有变化,而且在各暴露组中具有时间效应和剂量效应关系;血生化检测指标中LDH活性,CK、AST、ALT和sEPCR含量五项指标均有变化,在各暴露组中也同样存在时间效应和剂量效应关系。
3)氧化应激检测结果:检测血清中GSH和O2ˉ·水平,碳纳米管高剂量组GSH水平下降明显较其它处理组高,SiO2高剂量处理组O2ˉ·水平最高。总之,碳纳米管暴露组在相同的剂量和染毒时间条件下明显较空白对照组和阴性对照组高。
4)血清中细胞因子TNF-α和IL-1β检测结果:随着染毒剂量的增加和染毒时间的延长,血清中TNF-α和IL-1β水平呈现一定的剂量效应和时间效应关系。而且在碳纳米管高剂量暴露组含量最高。
5)血清中细胞粘附分子ICAM-1和VCAM-1检测结果:随着染毒剂量的增加和染毒时间的延长,血清中ICAM-1和VCAM-1水平呈现一定的剂量效应和时间效应关系。而且碳纳米管高剂量暴露组水平最高。
6)免疫组化实验结果:利用免疫组化方法对主动脉壁的突起部分进行ICAM-1和VCAM-1表达的检测,结果表明高剂量碳纳米管30d处理组主动脉壁上ICAM-1的表达强于VCAM-1 ,但二者均明显高于空白对照组。
2.细胞水平实验:将碳纳米管配制成由高到低依次为200、100、50、25、12.5、6.25、3.12μg/ml的悬液,分别染毒传代培养2-3代的大鼠主动脉血管内皮细胞,利用细胞培养液作为空白对照,测试指标与结果如下:
1)氧化应激:分别于染毒结束后检测细胞培养液上清中LDH活性、GSH水平。细胞内LDH活性在碳纳米管染毒浓度为0~100μg·mL-1范围内随染毒剂量的增加而逐渐上升,在200μg·mL-1剂量组,其释放有所减弱;而培养液上清中GSH在碳纳米管染毒浓度为0~200μg·mL-1范围内,其含量随染毒剂量的增加而逐渐下降。
2)细胞超微结构的改变:电镜扫描结果表明,碳纳米管能够穿透血管内皮细胞膜进入细胞质并在其中聚集,进而对细胞器产生损伤作用。在100μg·mL-1剂量暴露12h组表现内质网及线粒体略肿胀,细胞溶酶体增加,染色质边染,但细胞膜完整;而在暴露20h组内皮细胞形态不完整,大部分RAECs水肿,坏死、脱落基底膜裸露,有些区域甚至断裂,核皱缩;线粒体明显肿胀,形状不规则空泡变,外膜模糊,内嵴不清,基底致密等退行性变;小部分区域内内皮细胞较完整,病变轻,仅细胞内线粒体略肿胀,胞浆基质疏松变淡,线粒体空泡变性,粗面内质网扩张,其内脂滴增多,内皮细胞间连接处微绒毛增多。
3)细胞存活率的改变:利用MTT实验方法检测不同浓度碳纳米管暴露不同时间后,大鼠主动脉血管内皮细胞存活率的改变。结果表明,随着碳纳米管染毒浓度的上升和染毒时间的延长,大鼠主动脉血管内皮细胞存活率逐渐下降,死亡率逐渐上升。
4)单个核细胞-血管内皮细胞粘附:电镜下RAECs呈单层、紧密贴壁,呈爬行式生长。细胞边缘不整齐,表面不光滑,有丝状或薄翼状的突起。PBMCs呈圆球形,表面不光滑,呈绒毛状。电镜下可见RAECs通过表面的突起粘附PBMCs,PBMCs也通过表面的绒毛与RAECs粘附。观察到“间接系链”(secondary-tethering)现象,即粘附在RAECs表面的PBMCs之间也有相互连接,或者是粘附在RAECs表面的PBMCs作为支撑点粘附了其它的PBMCs,结果使得RAECs与PBMCs的粘附加强和扩大;而且随着染毒剂量的增加大鼠主动脉血管内皮细胞与单个核细胞的粘附率显著增加。
5)细胞粘附分子ICAM-1和VCAM-1蛋白及mRNA的表达测定:利用ELISA试剂盒检测不同时间和不同浓度碳纳米管暴露后细胞培养液上清中细胞粘附分子ICAM-1和VCAM-1蛋白的表达;利用RT-PCR技术测定不同时间和不同浓度碳纳米管暴露后细胞ICAM-1和VCAM-1mRNA的表达。结果表明随着碳纳米管暴露时间和暴露浓度的增加,ICAM-1和VCAM-1mRNA和蛋白的表达均呈增加趋势,具有一定的时间效应和剂量效应关系。
6) IκKα、IκBα检测:为了进一步验证碳纳米管暴露致大鼠主动脉血管内皮细胞损伤的NF-kB途径,我们利用免疫印迹技术对IKKα、IκBα及NF-κB的表达进行检测,结果表明随着碳纳米管暴露剂量的增加,IKKα及NF-κB的表达呈上升趋势,而IκBα的表达呈下降趋势。
7)NF-kB/P65的核移位表达:利用免疫细胞化学方法对核转录因子NF-kB/P65的核移位表达进行检测,碳纳米管暴露组明显高于空白对照组和N-乙酰水杨酸预处理组,说明碳纳米管是通过致细胞炎症反应而致细胞损伤,而且是通过激活NF-kB/P65途径进行的。
研究结论:
1.整体动物实验结果表明碳纳米管暴露可致大鼠主动脉血管壁损伤,机体出现明显的氧化应激和炎症反应,血中前炎症因子TNF-α和IL-1β及反映血管内皮细胞功能的可溶性细胞间粘附因子(sICAM-1)和可溶性血管细胞粘附因子(sVCAM-1)较空白对照组和阴性对照组均明显升高。
2.细胞水平实验结果表明,碳纳米管暴露可致大鼠主动脉血管内皮细胞损伤。表现为细胞超微结构的改变和细胞存活率的下降,特别是作为血管内皮细胞功能紊乱的评价指标ICAM-1和VCAM-1 ,碳纳米管暴露后其mRNA和蛋白均有过度的表达,利用免疫印迹技术和免疫组化技术检测与这两种粘附分子表达相关的核转录因子NF-κB相关蛋白也呈现过度表达。
3.激活NF-κB信号通路途径:细胞水平实验表明活性氧激活血管内皮细胞内的核转录因子κB,启动血管内皮细胞内核转录因子κB信号途径而增加血管内皮细胞表面细胞间粘附分子-1的表达,介导中性粒细胞与血管内皮细胞的牢固粘附;而整体动物实验则是通过CNTs暴露致机体产生活性氧从而激活细胞内核转录因子κB信号途径,同时活性氧释放到细胞外,刺激巨噬细胞和T淋巴细胞分泌TNFα和IL-1β,在TNFα、IL-1β与核转录因子κB的协同作用下,血管内皮细胞中粘附分子表达增加,诱导并加强中性粒细胞与血管内皮细胞的粘附。
The most attractive properties of nanomaterials for medical and technologic applications, including their small size, large surface area, and high reactivity, are also the main factors for their potential toxicity. Based on their inhalation studies with ambient ultrafine particles, it has been predicted that nanosized particles will have higher pulmonary deposition and biological activity compared with larger particles. Thus, some nanosized materials may induce not only damage at the deposition site but also distant responses as a result of their translocation and/or reactivity through the body. In this respect, epidemiologic and experimental studies have suggested an relation between respiratory exposure to ambient ultrafine particles ( including particles with a diameter <100nm ) and the progression of cardiovascular disease. Engineered carbon nanomaterials, including carbon nanotubes, have elicited a great deal of interest recently because of their unique electronic and mechnical properties. Carbon nanotubes(CNT ) have a diameter ranging from 0.7 to 1.5nm, with lengths >1μm. Initial toxicologic studies demonstrated that intratracheal or pharyngeal instillation of CNTs suspension in mice caused a persistent accumulation of carbon nanotube aggregates in the lung, followed by the rapid formation of pulmonary granulomatous and fibrobic tissues at the site. The unique physical characteristic and the pulmonary toxicity of CNTs raised concerns that respiratory exposure to these materials might be associated with systemic toxicities. In the present studies, we demonstrate that lung instillation of CNTs is associated with a dose-dependent increase in oxidative modifications, which are considered to play a role in atherogenesis, we further evaluated the effects of CNTs respiratory exposure on rat.
Since endothelium is the first layer of vascular, endothelial cells would be the target to be injured firstly. The cell adhesion molecules including ICAM-1 and VCAM-1 are important for binding of leukocytes to the endothelial cells and in the infiltration of inflammatory cells into tissues. Nuclear factor-kB (NF-kB) has been implicated in the transcriptional activation of the genes encoding CAMs. Rapid phosphorylation and degradation of IkBαallows NF-κB to translocate into the nucleus and to regulate transcription of the target genes. As very little is known in regard to the toxicology and the underlying mechanism involved in explaining the phenomena of carbon nanotubes (CNTs) exposure, we carried out a detailed study on their effects of the expression of cell adhesion molecules on rat aortic endothelial cells ( RAECs). We demonstrate here that CNTs induced adhesion of neutrophils to endothelial monolayer by promoting the expression of ICAM-1, VCAM-1. Since NF-κB is a major transcription factor involved in the transcriptional regulation of cell adhesion molecules, thus we studied the status of NF-κB/P65 activation in CNTs treated RAECs. As oxidative stress is known to regulate the activation of NF-κB, we tested GSH content and LDH activity. We found that CNTs induced oxidative stress and调the expression of ICAM-1 and VCAM-1. The nucleus translocation of NF-κB/P65 after CNTs exposure could be inhibited by N-acetylcysteine which indicated that the high expression of ICAM-1 and VCAM-1 mediated by oxidative stress in rat aortic endothelial cells might play an important inflammation role in CNTs-induced vascular endothelium damage. After establishing RAECs damage model, we measured the expression of ICAM-1 and VCAM-1 in cultured RAECs stimulated with different dose of CNTs for different time exposure and detected NF-κB signaling pathways.
Method :
we evaluated the toxic effects of intratracheal instillation of CNTs on rats and CNTs exposure on RAECs. The related indexes including oxidative damage, inflammatory factor, adhesive molecular and signal molecular were detected by biochemical assays, immunological histochemistry, RT-PCR and Western blot. Data analyzed by using SPSS1 .0 software.
Result:
1. Animal test:
Rats were intratracheally instilled with 0, 0.7, or 3.5 mg of carbon nanotubes, carbon black as negative control, or quartz as positive control. The rats were euthanized on 7 d or 30 d after the single treatment per day for study of histopathological changes in some organs and expression of sICAM-1 and sVCAM-1in rats’blood.
1)Histopathological examination:
Histopathological evaluation of lung tissues revealed that lung in rats exposed to CNTs and quartz particles produced a dose-dependent lung inflammatory response characterized by neutrophils infiltration and foamy (lipid-containing) alveolar macrophage accumulation. In addition, lung tissue thickening as a prelude to the development of fibrosis was evident and progressive. Histopathological evaluation of aortic vascular tissue revealed that CNTs exposure fior 30d resulted in a protuberance on inner membrane of aortic vascular, but CB and SiO2 groups had no this kind phenomenon.
2)Blood routine examination and blood biochemistry assay:
The results of blood routine examination showed that only the contents of PLT、WBC and ?b value were different between NS control group and CNTs、SiO2 experimental groups, especially in CNTs experimental group these index showed the highest value. The results of LDH activity, CK、ALT、AST and sEPCR contents increased in a dose dependant manner and time dependant manner under treatment with all particle types especially the highest release induced by SiO2 at a particle concentration of 17.5 mg/kg. However, maximal LDH release induced by CNTs treatments was noted to be lower than that observed with SiO2 (positive control) possibly due to high particle concentrations interfering with the assay.
3)Oxidative stress and formation of reactive oxygen species:
A significant depletion of GSH was observed at high dose of CNT exposure compared with control group, and was also time- dependent. But the content of O2ˉ·increased with the time and dose of CB、CNTs and SiO2 exposure, and a significant increase in CNTs and SiO2 exposure compared with control group. Overall, the results demonstrated a significant depletion of GSH and increase in oxidative damage levels occurred in CNTs exposed rats.
4)Inflammatory response (TNF-αand IL-1β):
Our results showed that CNTs exposure elevated the expression of TNF-αand IL-βin dose and time dependent manner, although there was only weak expression in low dose group.And in the case of the same dose treatment, the longer the CNTs exposure, the higher expression level of them occurred, and the expression level of these two inflammatory factors in rats exposed to CNTs exposure was greater than that of CB and SiO2 at the same dose and the same time.
5)sICAM-1 and sVCAM-1 expression of blood:
CNTs increased monocyte adhesion to cells dose-dependently and time-dependently. Meanwhile, CNTs exposure elevated the expression of sICAM-1 and sVCAM-1 in dose and time dependent manner, although there was only weak expression in low dose group.And in the case of the same dose treatment, the longer the CNTs exposure, the higher expression level of them occurred, and the expression level of these two adhesion molecular in rats exposed to CNTs was greater than that of CB and SiO2 at the same dose and the same time.
6)Immunohistochemistry of ICAM-1 and VCAM-1 on aortic vascular after CNT
exposure:
The results of immunohistochemistry showed that ICAM-1 and VCAM-1 only expressed infirmly in endothelial cells of NS control group , but VCAM-1 expressed positively in endothelial cells and protuberance of CNTs experiment group, which were located in cytoplasm, cytomembrane and intercells; ICAM-1 expressed firmly in protuberance including endothelial cells even smooth muscle cells of midmembrane.
2.Cell test:
RAECs were exposed to CNTs suspension at different dose and for different time.
1)In vitro cell oxidative stress: A significant depletion of GSH was observed at dose 50-200μg·mL-1 of CNTs compared with that in the control group. Overall, the results demonstrated a significant depletion of GSH levels occurred in RAECs exposed to CNTs. The results of LDH leakage and exhibited a significant cytotoxicity at CNTs concentrations of 12.5-100μg·mL-1. There was a statistically significant difference between different dose of CNTs, whereas the highest toxicity was at 100μg·mL-1 but not 200μg·mL-1.The detailed mechanism is not clear, which needs more work to do.
2)CNTs localization and ultrastructure observation: After exposure to CNTs, there were clear changes in the ultramicroscopic features of RAECs. CNTs frequently lodged inside cytoplasm and mitochondria. RAECs incubated with CNTs showed extensive disruption of mitochondrial cristae, resulting in a vacuolar cellular appearance and microvillus. These changes were time dependent, with fewer particles collecting inside mitochondria during shorter incubations. Electron microscopy showed considerable mitochondrial damage by CNTs, resulting in the formation of concentric structures, known as myelin figures.
3)Cell viability:
The MTT assay showed CNTs was toxic to RAECs and the cytotoxicity increased with CNTs doses and exposure time. From these results, we could see that most mitochondria were damaged characteristically by swelling with architectural disruption which was consistent with the qualitative analysis.
4)SEM image of adhesion between RAEC and PMBC after CNTs exposure:
Under the SEM, RAECs grew as a monolayer, adhering to culture bottle wall like creeping. Cells’border was not tidy, surface not smooth and had silk-like salience. RAECs could connect each other by these saliences. PBMC were like ball and their surfaces were not smooth which looked like microvillus. RAECs adhered to PMBCs through these saliences under SEM. PMBCs could also adhere to RAECs by microvillus on their surfaces. Secondary-tethering phenomenon could be observed: connection between PMBCs which jointed to the surfaces of RAECs or backstop adhered to the surface of RAECs provided a point to adhere to other PBMCs which led to strengthen and enlarge adhesion between RAECs and PMBCs. The degree of adhesion increased with the doses of CNTs.
5)Expression of ICAM-1 and VCAM-1 protein and mRNA:
CNTs exposure elevated the expressions of ICAM-1 and VCAM-1 in dose and time dependent manner.By one way analysis of variance,the expression level of ICAM-1 and VCAM-1 in vascular cellular endothelium in high dose groups (25-200ug·mL-1) was statistical higher than that in the control group.And in the case of the same dose treatment, the longer the CNTs exposure, the higher expression level of them occurred, but the difference of expression level between exposure for 12h and 24h was greater than that for 24h and 48h. In agreement with the protein data, noticeable changes were detected in the steady-state mRNA levels of both ICAM-1 and VCAM-1.
6) CNTs exposure induced activation of NF-κB, IKKαand the degradation of IκBα:
Comparison of the levels of NF-κB in the nuclear fraction indicated that the level of NF-κB in the nuclear fraction was higher in CNTs exposure cells than that in the control cells. Western blotting and densitometric analysis of the density of bands relative toβ-actin indicated that CNTs exposure resulted in markedly greater activation of NF-κB and its translocation to the nucleus than that in control RAECs. However, the CNTs induced activation and translocation of NF-κB to the nucleus in the RAECs was greater than that in the control RAECs; Western blot analysis and subsequent measurement of density of bands relative toβ-actin indicated that activation level of IKKαwas higher in CNTs exposure RAECs than that in the control RAECs. Greater degradation of IκBαwas noted in CNTs exposure RAECs as compared to the control RAECs.
7)Effects of N- acetylcystein on nuclear translocated NF-κB/p65 induced by CNTs in RAECs:
The immunoanalysis of effects of arecholine on nuclear translocated NF-κB/p65 induced by CNTs in RAECs showed that CNTs exposure increased the nuclear translocated NF-κB/p65 compared with that in the control group, however pretreating the cells with N-acetylcystein before CNTs exposure, the nuclear translocated NF-κB/p65 decreased but was still higher than that in the control group. It suggested that N-acetylcysteine could inhibit CNTs to induce NF-κB/p65 expression on rat aortic endothelial cells. ROS production and oxidative stress were probably some of the mechanisms.
Conclusion:
1. An important consideration is that oxidative stress, and especially depletion of reduced GSH and O2ˉ·production, the leakage of IL-1βand TNF-αincreasing with dose and time of CNTs exposure, which is the same as the results of sICAM-1 and sVCAM-1. These results show that, for the test conditions described here and on an equal-weight basis, carbon nanotubes can pass through lung-blood barrier and reach the blood, they are much more toxic than carbon black and can be more toxic than quartz, which is considered a serious occupational health hazard in chronic inhalation exposures. As markers of endothelial cells damage, the three indicators of experimental animals especially CNT group are higher than those of control group. In this study, we have sought to bring these findings to suggest pathobiologic processes whereby nanaoparticles, especially CNTs, had effects on the cardiovascular system. Pathologic end points relevant to plaque rupture, endothelial erosion, hemostasis, and coagulation should be used in toxicologic studies.
2. The results of cell tests showed that CNTs exposure can induce RAECs damage which including ultrastructure of RAECs changed and cell viability decreased, especially the expression of ICAM-1 and VCAM-1 mRNA and protein are higher than that in control RAECs.
3. We demonstrate here that CNTs induce adhesion of neutrophils to endothelial monolayer by promoting the expression of ICAM-1, VCAM-1. Since NF-κB is a major transcription factor involved in the transcriptional regulation of cell adhesion molecules, thus we studied the status of NF-κB/P65 activation in CNTs treated RAECs. As oxidative stress is known to regulate the activation of NF-κB, we tested GSH content and LDH activity. We found that CNTs induced oxidative stress and the expression of ICAM-1 and VCAM-1. The nucleus translocation of NF-κB/P65 after CNTs exposure can be inhibited by N-acetylcysteine, which indicate that the high expression of ICAM-1 and VCAM-1 mediated by oxidative stress in rat aortic endothelial cells may play an important inflammation role in CNT-induced vascular endothelium damage.
引文
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2.Pantarotto D, Briand JP, Prato M, et al. Translocation of bioactive peptides across cell membranes by carbon nanotubes. Chem Commun, 2004,1:16-17.
3．Kam NWS, Jessop TC, Wender PA, et al. Nanotube molecular transporters: internalization of carbon nanotube-protein conjugates into mammalian cells. J Am Chem Soc, 2004,126(22): 6850-6851.
4．Donaldson K, Li XY, Dogra S, et al. Asbestosstimulated tumor necrosis factor release from alveolar macroPhages depends on fiber length and opsonization. J Pathol, 1992, 168(2): 243-248.
5．Lam CW, James JT, McCluskey R, et al. Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation. Toxicol Sci, 2004, 77: 126-134.
6．Muller J, Huaux F, Mercer R. et al. Respiratory toxicity of multi-wall carbon nanotubes. Toxicol Appl Pharmacol, 2005,207(3):221-231.
7．Huczko A, Lange H, Bystrzejewski M, et al. Pulmonary toxicity of 1-D nanocarbon materials. Fullerenes Nanotubes Carbon Nanostruct, 2005,13(2):141-145.
8. Gerlofs-Nijland ME, Boere AJF, Leseman DL, et al. Effects of particulate matter on the pulmonary and vascular system: time course in spontaneously hypertensive rats. Particle Fibre Toxicol, 2005, 2:2.
9. Dockrey DW, PoPe CA, Xu X, et a1. An association between air pollution and mortality in six U S cities N Engl J Med, 1994, 329:1753—1759.
10.PoPe C A, Hill R W, Villegas GM. Particulate air pollution and daily mortality on Utah's Wasatch Front[J]. Environ Health Perspect, 1999, 107: 567 – 573.
11.Yamawa H, Iwai N. Mechanisms underlying nanosized air pollution mediated progression of atherosclerosis：carbon black causes cytotoxic injury/inflammation and inhibits cell growth in vascular endothelial cells. Circulation, 2006, 70(1): 129-140.
12. Penttinen P, Timonen KL, Tiittanen P, et al. Ultrafine particles in urban air and respiratory health among adult asthmatics. Eur Resp , 2001, 17: 428 – 435.
13. Donaldson K, Tran L, Jimenez LJ, Combustion-derived nanoparticles: A review of their toxicology following inhalation exposure. Particle Fibre Toxicol, 2005, 2:10.
14. Barlow PG, Clouter-Baker A, Donaldson K, Carbon black nanoparticles induce tyPe II epithelial cells to release chemotaxins for alveolar macrophages. Particle Fibre Toxicol, 2005,2:11.
15. Oberosters G, Urell MJ. Ultrafine particles in the urban air: to the respiratory tract and beyond? Environ Health Perspect, 2002, 110(8): 440-441.
16. Renwick LC, Brown D, Clouter A, et al. Increased inflammation and altered macrophage chemotactic response caused by two ultrafine particle types. Occup Environ Medi. 2004, 61(5):442-447.
17. Nakada Y, Fattal E, Foulquier M, et al. Pharmacokinetics and biodistribution of oligonucleotide adsorbed onto poly ( isobutylcyanoacrylate ) nanoparticles after intravenous administration in mice. Pharm Res, 1996, 13(1): 38-43.
18. Godard G, Boutorine AS, Saison BE, et al. Antisense effects of cholesterol-oilgodeoxynucleotide conjugates associated with Poly (alkylcyanoacrylate) nanoparticles. Eur JBiochem, 1995, 232(2): 404-410.
19. Hwang SJ, Bellocq NC, Davis ME. Effects of structure of β-cyclodextrin-containing polymers on gene delivery. Bioconjug Chem, 2001,12(2): 280-290.
20. Kwok KY, Adami RC, Hester KC, et al. Strategies for maintaining the particle size of peptide DNA condensates following freeze-drying. Int J Pharm, 2000, 203(1-2): 81-88.
21. Lukacs GL, Haggie P, Seksek O, et al. Size-dependent DNA mobility in cytoplasm and nucleus. J Biol Chem, 2000, 275(3): 1625-1629.
22. Brunner S, Sauer T, Carotta S, et al. Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther, 2000, 7(5): 401-407.
23. Wightman L, Kircheis R, R?ssler V, et al. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med, 2001, 3(4): 362-372.
24. Kaur S, Clark RDR, Walsh PT, et al. Exposure visualition of ultrafine particle counts in a transport microenvironment. Atmos Environ, 2006, 40: 386-398.
25. Peters A, Dockery DW, Muller JE, et al. Increased particulate air pollution and the triggering of myocardial infarction. Circulation, 2001, 3: 2810-2815.
26. Hartog JJ., Hoek G., Peters A, et al. Effects of fine and ultrafine particles on cardiorepiratory symptoms in elderly subjects with coronary heart disease: the ULTRA study. Am J Epidemiol, 2003, 157: 613-623.
27. Loft S, Risom L, Moller P. Chemical composition and surface areas in relation to health effects of ultrafine particles-reply to Ovrevik and Schwarze. Mutat Res. 2006, 594: 199-200.
28. Ibald-Mulli A., Timonen K. L., Peters A., et al. Effects of particulate air pollution on blood pressure and heart rate in subjects with cardiovascular: a multi-center approach. Environ Health Perspect, 2004, 112: 369-377.
29. Kim CS, Kang TC. Comparative measurement of lung deposition of inhaled fine particles in normal subjects and patients with obstructive airway disease. Am J Respir Crit Care Med, 1997,155:899-905.
30. Sunyer J,Schwartz J,Tobias A, et al. Patients with chronic obstructive pulmonary diseaseare at increased risk of death associated with urban particle air pollution: a case-crossover analysis. Am J Epidemiol, 2000, 151: 50-56.
31. Schins RPF, Lightbody JH, Borm PJA, et al. Inflammatory effects of coarse and fineparticulate matter in relation to chemical and biological constituents. Toxicol appl Pharmacol, 2004, 195: 1-11.
32. Donaldson K, Li XY, MacNee W. Ultrafine (nanometre) particle mediated lung injury. JAerosol Sci, 1998, 29: 553-560.
33. Suwa T, Hogg JC, Quinlan KB, et al. Particulate air pollution induces progression of atherosclerosis. J Am Coll Cardiol. 2002 Mar 20;39(6):935-42
34. Nemmar A, Hoet PH, Vermylen J, et al. Pharmacological stabilization of mast cells abrogates late thrombotic events induced by diesel exhaust particles in hamsters.Circulation. 2004 Sep 21;110(12): 1670-7
35. Radomski A, Jurasz P, Alonso-Escolano D, et al. Nanoparticle-induced platelet aggregation and vascular thrombosis. Br J Pharmacol. 2005 Nov;146(6):882-93
36. Gilmour PS, Ziesenis A, Morrison ER, et al. Pulmonary and systemic effects of short-term inhalation exposure to ultrafine carbon black particles.Toxicol Appl Pharmacol. 2004,195(1):35-44
37. Coultas L, Chawengsaksophak K, Rossant J. Endothelial cells and VEGF in vascular development. Nature, 2005, 438(7070): 937-945.
38. Masuda M, Takahashi H. Adhesion of leukocytes to endothelial cells in atherosclerosis. RinshoByori, 1998,46(11): 1149-1155.
39. Vander Wal AC, Becker AE, Das PK. The expression of adhesion molecules on endothelial cells in atherosclerosis has a topographic relationship with the underlying inflammatory process. Histopathology, 1994, 24(2): 200-201.
40. Wang X, Chen J, Tao Q, Zhu J, Shang Y. Effects of ox-LDL on number and activity of circulating endothelial progenitor cells. Drug Chem Toxicol, 2004, 27(3): 243-255.
41. 刘达, 姚智. NF-κB及其活化的研究进展. 天津医科大学学报, 2001, 1(7): 139-142.
42. Delfino RJ, Sioutas C, Malik S. Potential role of ultrafine particles in associationsbetween airborne Particle mass and cardiovascular health. Environ Health Prospect , 2005,113(8): 934-946.
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