【作者】 徐玉英；

【导师】 徐立红； 朱心强；

【作者基本信息】 浙江大学 ， 劳动卫生与环境卫生学， 2011， 博士

【摘要】 背景心血管疾病是全球范围内造成死亡的最主要原因。大量流行病学研究表明,心血管疾病与空气颗粒(particulate matter, PM)污染物暴露有关。各种PM对心血管系统损伤很大,而且颗粒物体积越小,危害性越大。多壁碳纳米管(MWCNTs)是属于纳米级颗粒,主要应用于纳米线圈、电子元件、催化剂等,并逐渐发展到药物载体等生物医药领域,其心血管暴露的机会增加,但目前对其生物安全性评价主要集中于肺毒性的研究。研究表明MWCNTs诱发肺部和/或全身的炎症反应,并使血管血红素氧合酶(heme oxygenase-1, HO-1)基因活化及氧化损伤,可能引起血管内皮功能损伤,形成前凝聚状态,具有一定的心血管系统毒性效应。内皮损伤是血管病理改变的主要早期事件,最终可引起一系列心血管相关的疾病。我们前期的研究表明,MWCNTs可引起内皮细胞发生DNA损伤和凋亡。本课题用体内外实验相结合的方法,研究MWCNTs对内皮损伤的机制,为后期的实验奠定基础。在明确MWCNTs对内皮的损伤作用之后,采用SD大鼠高脂饮食诱发动脉粥样硬化模型,观察MWCNTs的心血管系统毒性。考虑到肺部炎症和脂肪肝程度都与心血管系统密切相关,所以,除了观察心脏、主动脉和血液,还有肺脏和肝脏。为了进一步验证MWCNTs对AS的影响,并且比较其他碳纳米材料的影响程度,本项目采用典型的高脂饮食LDLR-1小鼠高脂饮食诱发高脂血症模型,比较研究了单壁碳纳米管(SWCNTs)、MWCNTs和多壁碳纳米圈(MWCNOs)的心血管毒性。目的、方法和结果1为了研究MWCNTs对内皮细胞结构和功能的改变情况,进行了体内实验和体外实验。其中体内实验把1%Tween 80作为溶剂对照组,200μg/kg单壁碳纳米管和1 mg/kg脂多糖作为阳性对照组,观察200μg/kg MWCNTs连续静脉注射7 d后,利用免疫组化和western blot测定内皮细胞vWF的表达情况；用ELISA测定血浆中的vWF和sICAM-1,综合评价内皮细胞的损伤情况。体外实验把完全培养液作为溶剂对照组,观察各浓度的MWCNTs对人脐静脉内皮细胞(HUVECs)作用不同时间后,利用电阻仪测定内皮细胞的电阻,western blot测定内皮细胞ZO-1的表达情况,评价内皮细胞的功能性损伤；利用台盼蓝试验测定细胞活力；western blot测定内皮细胞procaspase-3、活化的caspase-3、p62和Atg8蛋白,评价内皮细胞损伤的机制。结果表明：200μg/kg MWCNTs连续静脉注射SD大鼠7d后,内皮细胞vWF表达增加,并分泌进入血液,造成内皮功能性和结构性损伤,导致血液中sICAM-1水平降低(P<0.05)；不同浓度的MWCNTs作用于人脐静脉内皮细胞(HUVECs) 0-48h, TEER降低,并有时间和浓度依赖效应；不同浓度的MWCNTs处理24 h后,ZO-1蛋白表达降低,内皮细胞的通透性增加(P<0.05),细胞活力降低,procaspase-3、活化的caspase-3、p62三个蛋白表达降低,而LC3-Ⅰ/LC3-Ⅱ的转化升高(P<0.05)。2为了研究50、100和200μg/kg多壁碳纳米管(MWCNTs)连续处理高脂饮食SD大鼠1-4个月后对血液、心、主动脉、肝和肺的影响,HE染色观察心、主动脉、肝和肺部的病理情况；油红O染色和茜素红染色分别观察主动脉内膜脂质沉积和主动脉钙化；用各个试剂盒测定血清和肝中的CHOL、TG和HDL-c,反映血脂和肝中脂类的情况,测定血清和肝中的GSH、MDA和T-SOD,反映氧化情况,测定血清和肝中的GOT、GPT和γ-GT,反映肝功；荧光定量PCR测定肺部炎症因子IL-1β和TNF-α的mRNA水平。结果表明：MWCNTs升高血清中的TG水平；50、100和200μg/kg MWCNTs作用2-4个月,主动脉内膜脂质沉积面积增加,肺间隔不同程度增厚,形成大小不同的肺肉芽肿块,GOT、GPT、γ-GT、GSH、T-SOD和MDA受到不同程度影响,肝中的脂类发生变化；50、100、200μg/kg MWCNTs作用2或4个月,主动脉钙化程度加重。3进一步验证MWCNTs对动脉粥样硬化的影响,并与SWCNTs和MWCNOs进行比较。分别用SWCNTs(2 mg/kg)、MWCNTs(2 mg/kg)和MWCNOs(20 mg/kg)分别处理高脂饮食LDLR-1小鼠3个月后,用HE染色观察心、主动脉、肺、肝和脾的病理情况；用各个试剂盒测定血清中的CHOL、TG、LDL-c和HDL-c,反映血脂的情况；用各个试剂盒测定血清中的GSH、MDA和T-SOD,反映氧化情况；用各个试剂盒测定血清中的GOT、GPT和y-GT,反映肝功。结果表明：SWCNTs、MWCNTs和MWCNOs都使肺间隔增厚,肺部肉芽肿形成,但MWCNTs在肺部蓄积更多,使肺脏系数增大(P<0.01),形成更多更大的肉芽肿；MWCNTs和MWCNOs使心脏系数和脾脏系数明显降低；SWCNTs、MWCNTs和MWCNOs都使血脂降低,主动脉脂肪变程度降低；SWCNTs、MWCNTs和MWCNOs对脂肪肝病变的影响并不显著；SWCNTs、MWCNTs和MWCNOs使血清中的GSH下降的幅度不同；MWCNTs和MWCNOs使肝功受到影响。结论1多壁碳纳米管使内皮发生功能性和结构性损坏。2多壁碳纳米管加重动脉粥样硬化。3单壁碳纳米管、多壁碳纳米管和多壁碳纳米圈都降低血脂。更多还原

【Abstract】 BackgroudCardiovascular diseases are the world’s largest killers. Numerous epidemiologic studies have shown that increased levels of environmental particulate matter (PM) pollutants are positively associated with cardiovascular diseases. PM exposure may trigger serious cardiovascular disorders, and the smaller particle volumes, the greater harmfulness. Multi-walled carbon nanotubes (MWCNTs) are a kind of nano-particles. They are used in various applications, such as nano-coil, electronic parts, catalysts, and even in medicine field. The chance of CNT exposure to cardiovascular system is increased. However their biosafety evaluation mainly focuses on pulmonary toxicity. A lot of studies showed that MWCNTs induced lung and/or systemic inflammatory response, and activated hemeoxygenase gene and induced oxidative damage. They may cause endothelial dysfunction, forming condensed state of blood. MWCNTs have a certain toxic effects of cardiovascular system. Vascular endothelial injury is the major early event in the pathological changes, and ultimately leads to a series of cardiovascular diseases. Our previous studies showed that MWCNTs induced DNA damage and apoptosis in endothelial cells. So in this study in vivo and in vitro tests were employed to explain the mechanism of endothelial injury, and laied the foundation for the later experiments.After it is clear that MWCNTs can induce endothelial damge, we investigated the cardiovascular toxicity of MWCNTs using high-lipid-diet SD rats. Taking into account the degree of lung inflammation and fatty liver closely related with cardiovascular system, we also observe lung and liver in addition to observation of heart, aorta and blood.To further verify the impact of MWCNTs on atheroclerosis, and compared with other nanocarbons, we use LDLR-/-mice with high fat diet as a model, to comparative study of SWCNTs, MWCNTs and MWCNOs.Aims, methods and results1 To study on endothelial damage including functional and organic changes induced by multi-walled carbon nanotubes (MWCNTs), using immunohistochemistry and western blot to detect the expression of vWF in endothelial cells, and using ELISA to detect plasma vWF and sICAM-1 are to comprehensive evaluation of organic damage of endothelial cells; using resistance tester to detect the resistance of endothelial cells, and using western blot to detect the expression of ZO-1 in endothelial cells are to evaluate the functional damage of endothelial cells; using western blot to detect the expression of procaspase-3, cleaved caspase-3, p62 and atg8 are to explain the molecular mechanism. We found that, after continuous intravenous inject 200μg/kg MWCNTs 7d, vWF expression was increased in endothelial cells and secreted into blood, resulting in injury of endothelial function, leading to sICAM-1 decreased in serum (P＜0.05). Different concentrations of MWCNTs treated HUVECs 0-48 h, the cell resistance is time and concentration-dependent lowered. After MWCNTs treated 24 h, ZO-1 expression decreased, endothelial cell permeability increased (P＜0.05). Different concentrations of MWCNTs treated HUVECs 24 h, cell viability decreased, procaspase-3, cleaved caspase-3, and p62 expression decreased, vWF and atg 8 expression increased (P＜0.05). 2 After different doses of multi-walled carbon nanotubes treated 1-4 months, observing the influence on blood, heart, aorta, liver and lung. Using HE staining to observe heart, aorta, lung, liver and spleen pathology. Using each kit to detect GSH, MDA, or T-SOD in serum or liver, which reflect the oxidative situation. Using each kit to detect GOT, GPT, orγ-GT in serum or liver, which reflect liver function. Using oil red O staining and alizarin red staining to observe aortic lipid deposition and aortic calcification. Using fluorescence quantitative PCR to detect plumonary inflammatory cytokines IL-1βand TNF-αmRNA levels. They are shown that MWCNTs elevated serum TG levels.50,100,200μg/kg MWCNTs treated rats after 2-4 months increased aortic intimal lipid depositon areas.50,100,200μg/kg MWCNTs treated rats after 2 or 4 months exacerated the degree of aortic calcification.50,100,200μg/kg MWCNTs treated rats after 2-4 months made different influence on GOT, GPT,γ-GT, GSH, T-SOD, MDA level.50,100,200μg/kg MWCNTs treated rats after 2-4 months changed the lipid in the liver.50,100, 200μg/kg MWCNTs treated rats after 2-4 months made lung septal thickening differently, and form pulmonary granuloma in different sizes.3 To comparison of single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), and multi-walled carbon nano-onions (MWCNOs) to observe the different influences on blood, heart, aorta, lung, liver and spleen in high-lipid-diet LDLR-/-mice. Using HE staining to observe heart, aorta, lung, liver and spleen pathology.Using each kit to detect serum CHOL, TG, LDL-c or HDL-c, which were reflecting the situation of blood-fat. Using each kit to detect serum GSH, MDA, or T-SOD, which reflect the oxidative situation. Using each kit to detect serum GOT, GPT, orγ-GT, which reflect liver function. They are shown that SWCNTs, MWCNTs or MWCNOs induced lung septal thichening and pulmonary granuloma formation. However, MWCNTs had more accumulation in the lung, and increased the lung index, and then form more and bigger granulomas. The heart index and spleen index were significantly decreased by MWCNTs or MWCNOs. SWCNTs, MWCNTs, or MWCNOs decreased blood fat, and the extent of aortic fatty degeneration. The impact of fatty liver, induced by SWCNTs, MWCNTs or MWCNOs, was no significant difference. SWCNTs, MWCNTs or MWCNOs decreased GSH level in serum. The liver functions are affected by MWCNTs or MWCNOs.Conclusion1. MWCNTs induced functional and structural damage in endothelial cells.2. MWCNTs aggregated atherosclerosis.3. SWCNTs, MWCNTs or MWCNOs can be lower blood lipids.更多还原