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题名: 纳米银诱导线粒体损伤及肝细胞毒性
作者: 王丰邦
学位类别: 硕士
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: 宋茂勇
关键词: 纳米银,肝细胞,线粒体损伤,细胞凋亡 ; silver nanoparticles hepatocyte mitochondrial lesion apoptosis
其他题名: The mitochondrial lesion and hepatocellular toxicity induced by nanosilver
学位专业: 环境工程
中文摘要:       纳米银(nAg)优异的物理化学性质和抗菌性能使其广泛地应用于医疗、食品和生物等行业中。近年来随着商业化纳米银产品种类和产量的显著增加,nAg释放到环境并进入人体的环境与健康风险不断加大,其安全性引起了人们广泛的关注。许多体内研究表明肝脏是纳米银主要累积器官之一。为研究nAg对肝细胞的毒性效应和作用机制,我们选择20 nm和100 nm的纳米银颗粒(nAg-20和nAg-100)和Ag+对肝癌细胞HepG2和肝正常细胞LO2进行暴露,以线粒体为主要研究对象,研究了不同粒径nAg和Ag+对肝细胞的毒性效应差异及作用机理。
      我们研究了细胞活性、细胞膜完整性、细胞凋亡、ROS水平与nAg暴露剂量效应关系;通过ICP-MS检测了细胞摄入总银水平,采用高分辨TEM观察了两种粒径nAg在细胞中的分布状况;最后,以线粒体为研究重点,考察了nAg对细胞线粒体超氧化物水平、线粒体膜电势、线粒体膜孔通透性、细胞色素C释放量及促凋亡酶caspase 3/7活性的影响。研究表明,2 μg/mL nAg和1 μg/mL Ag+是亚毒性剂量,此剂量可以诱导细胞出现凋亡,但细胞活性仍维持在80%以上;通过对细胞膜完整性检测发现,只有nAg-100和Ag+对HepG2细胞的膜完整性产生明显损伤,表明细胞膜损伤只是nAg产生细胞毒性效应的一部分;对细胞摄入nAg和Ag+量检测结果表明两种细胞摄入nAg-100的量基本相同,但对于nAg-20和Ag+,HepG2细胞摄入量大于LO2细胞,同时TEM观察细胞切片也证实了细胞摄入的nAg以粒子的形式存在;对线粒体状态研究表明,nAg和Ag+引起线粒体超氧化物上升并降低线粒体膜电势,但只有nAg颗粒可以引起细胞线粒体膜孔打开,释放出细胞色素C,激活下游凋亡酶caspase 3/7活性。
      总之,我们研究的结果表明nAg可以通过作用于线粒体诱导肝细胞毒性效应。在此作用过程中,nAg主要以粒子形式发挥效应,但其作用机制仍有待进一步研究。
英文摘要:       Silver nanomaterials are widely used in numerous fields like medical therapy, food, and biology due to its physiochemical and antibacterial properties. With the mounting number of commercialized nanosilver products, the potential risk of releasing silver particle from the products into environment and human beings has attracted huge attentions in recent years. Many in vivo studies have confirmed that silver nanoparticles (Ag NPs) could accumulate in liver and induce liver damage, but the underlying mechanism remains unclear. To investigate the difference of Ag NPs and Ag+ in cellular toxicity and the related mechanism, the 20 nm and 100 nm Ag NPs (nAg-20 and nAg-100) are exposed to hepatocellular carcinoma (HepG2) and normal hepatocyte (LO2) cell lines. More importantly, the effects of Ag NPs and Ag+ on mitochondria were investigated.
      Firstly, the cytotoxicity was assessed by cell viability, apoptosis and ROS level. And the cellular membrane integrity was also measured by the LDH releasing level. Then, we measured the total Ag in cell by ICP-MS and observed the cellular Ag NPs distribution by TEM. Lastly, we investigated cellular mitochondrial ROS, mitochondrial membrane potential (MMP), mitochondrial transition pore permeability (MTP), cytochrome C releasing and caspase 3/7 activity to assess the mitochondrial health and its role in cellular apoptosis affected by the selected dose. The results showed that 2 μg/mL Ag NPs and 1 μg/mL Ag+ are sub-lethal dose where the apoptosis emerged but the cell viability are beyond 80 %. Only nAg-100 and Ag + compromised HepG2 cell membrane integrity obviously, which implies the difference of cytotoxicity between Ag NPs and Ag+. The totally cellular Ag level indicated that the uptake of nAg-100 in the two cell lines are almost same, but nAg-20 and Ag+ level are distinctly higher in HepG2 than in LO2. TEM images confirmed that Ag NPs were still nanoparticles inside the both cell lines. We also found that both Ag NPs and Ag+ could stimulate mitochondrial ROS and decrease the MMP, but only Ag NPs incurred MTP opening, cytochrome C releasing and then increased the caspase 3/7 activity.
      Altogether, we found that Ag NPs could induce cytotoxicity of liver cell by inducing mitochondrial lesion. And the nanoparticles play an important role in this process. However, the mechanism still need to be further studied.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/36970
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
王丰邦. 纳米银诱导线粒体损伤及肝细胞毒性[D]. 北京. 中国科学院研究生院. 2016.
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