碲化镉量子点对原代肝、肾细胞及氧化应激蛋白质的毒性研究
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摘要
量子点(Quantum dots, QDs)是由IIB-VIA族元素(如CdSe, CdTe等)或IIIA-VA族元素(如InP, GaAs等)组成的粒径范围从2nm至100nm的半导体纳米晶体。与传统有机荧光材料相比,量子点因其具有发射光谱窄而对称、发射波长可调、激发光谱宽、抗光漂白性强等独特的光学特性,量子点在用于荧光探针、荧光共振能量转移中作为能量给体以及作为标记物进行光学成像等方面展现出巨大潜力。由于量子点中含有重金属元素,研究者也逐渐意识到量子点可能存在的潜在危害。研究表明肝脏以及肾脏是含镉量子点的主要毒性靶器官,也是体内镉的主要蓄积部位及靶器官。肝细胞常被用作评价镉的细胞毒性,肾小管上皮细胞是研究肾毒性的模式细胞。本文中我们选择原代肝细胞以及肾小管上皮细胞研究了水相合成的CdTe量子点的细胞毒性。此外,研究发现某些纳米材料在生物介质中会首先与生物大分子结合。这种结合赋予纳米粒子新的“生物识别身份”并决定着其后细胞或者组织器官的反应,所以有必要对量子点与其毒性作用发挥相关蛋白质(尤其是氧化应激蛋白质)的相互作用进行研究。本文从细胞水平和分子水平上探讨了CdTe量子点的细胞毒性效应。主要研究内容包括以下几个方面：
首先,我们利用“一锅法”合成了谷胱甘肽包被的CdTe量子点,并对合成的CdTe量子点进行了表征。
随后,利用CCK-8法初步分析了CdTe量子点的细胞毒性,从整体上评价了CdTe量子点对小鼠肾小管上皮细胞及肝细胞活力的影响。在此基础上主要利用流式细胞术探讨了经量子点暴露后含量子点及镉离子的细胞比例与量子点细胞毒性的关系、细胞对CdTe量子点所引发的氧化应激反应(RC、MDA含量以及过氧化氢酶、SOD的活性指标)以及氧化应激失效导致的细胞凋亡。另外,通过单细胞凝胶电泳技术研究了CdTe量子点产生的基因毒性。得出的主要结论有：(1)CdTe量子点对这两种细胞均产生了细胞毒性。肝细胞对CdTe量子点的耐受性要高于肾小管上皮细胞。(2)CdTe量子点能够进入两种细胞内。经CdTe量子点染毒后仅少数细胞内含Cd2+。随着染毒浓度的增大,含量子点的细胞数目不断增加。倒置荧光显微镜下的观察也证实了量子点在细胞内的存在。(3)CdTe量子点在细胞内释放出镉离子。随着暴露浓度的增大,含Cd2+的细胞比例呈逐渐上升趋势。细胞内Cd2+浓度的上升与CCK-8测得的细胞活力下降呈现出一定的相关性。“木马”效应解释了细胞内存在镉离子的现象。(4)CdTe量子点诱发细胞产生氧化应激效应。细胞内ROS产生量随着CdTe量子点暴露浓度的增加而增加。对两种细胞内与氧化应激相关酶的研究发现SOD的活力上升,而过氧化氢酶的活力出现下降。(5)CdTe量子点诱导细胞凋亡。经CdTe量子点暴露后,小鼠肾小管上皮细胞及肝细胞中处于凋亡期的细胞占比出现上升。(6)CdTe量子点造成细胞DNA断裂损伤。Olive尾距及尾部DNA含量指标随着CdTe量子点暴露浓度的增加均呈现明显上升趋势。
之后,运用光谱学及等温滴定量热技术等方法分别研究了谷胱甘肽包被的CdTe量子点与血清白蛋白、过氧化氢酶以及Cu/Zn-SOD的相互作用。研究结果表明CdTe量子点与这三种蛋白的相互作用较弱,未改变蛋白的结构。疏水力是CdTe量子点与这三种蛋白相互作用过程中的主要驱动力。对过氧化氢酶以及SOD的活性分析显示CdTe量子点对这两种抗氧化酶的功能没有产生影响。
基于流式细胞术、光谱学及等温滴定量热等实验技术,本文从细胞及分子水平对水相合成的CdTe量子点的毒性进行了探讨,研究结果有助于设计及合成生物相容性及稳定性更佳的量子点,并为合理利用并最大限度地降低量子点的负面效应提供了理论参考。
Quantum dots are group IIB-VIA (eg, CdSe, CdTe) or group IIIA-VA (eg, InP, GaAs) semiconductor nanocrystals in the size range of2-100nm. Due to their symmetric and narrow emission spectrum, tunable fluorescence emission, broad excitation spectra and high resistance to photobleaching, quantum dots have superior optical properties over other organic fluorescent dyes, and present a broad potential for biomedical applications. With the development of quantum dots applications, chance of people exposed to such substances is growing. Numerous studies have shown that liver and kidney are the main target organs for cadmium-based quantum dots, which are also found for cadmium. Hepatocytes are widely used for evaluation of cytotoxicity of cadmium, and tubular epithelial cells constitute an established model in renal toxicology. Therefore, primary hepatocytes and renal tubular epithelial cells were used in this research as an evaluation model for cytotoxicity of CdTe QDs. In addition, nanomaterials may be covered with a 'corona' of biological molecules like proteins immediately upon contact with the physiological environment. This combination gives these nanoparticles new "biological identity" and subsequently determine the response of cells or organs, hence we also investigated the interactions of CdTe QDs and proteins, which related to the cytotoxicity of quantum dots. In this research, we explored the toxicity of CdTe QDs from the cellular and molecular perspectives. The major works and results are as follows:
Firstly, glutathione-capped CdTe QDs were synthesized via a one pot method, and the characterization of the CdTe QDs was presented.
The cytotoxicity of CdTe QDs were preliminary analysed by assessment of cell viability was performed using the Cell Counting Kit-8(CCK-8) assay. To investigate cytotoxicity mechanisms of CdTe QDs, the cells containing CdTe QDs and cadmium ion, ROS content, cell oxidative damage (MDA content, the activity of catalase and SOD), cell apoptosis were determined. Moreover, single cell gel electrophoresis assay was employed to study the genotoxicity of CdTe QDs. The experimental results showed that:(1) CdTe QDs causes cytotoxicity to these two types of cells. CdTe QDs induced more serious cytotoxicity injury on renal tubular epithelial cells than hepatocytes.(2) CdTe QDs can enter these two cells. Only a small number of cells were found to contain Cd2+. The number of cells containing CdTe QDs increased with the exposure concentration. The observation from inverted fluorescence microscope confirmed the presence of the QDs in the cells.(3) Cd2+was released from the CdTe QDs after internalization in cells. The number of cells containing Cd2+increased with the exposure concentration. Trojan horse-type mechanism explained the release of cadmium ions in the renal tubular epithelial cells and hepatocytes.(4) CdTe QDs induced oxidative damage to these two types of cells. Cellular ROS content increased with the exposure dose; MDA content and the activity of SOD increased synchronous with QDs concentrations, while the activity of catalase declined.(5) The CdTe QDs induced apoptosis in renal tubular epithelial cells and hepatocytes.(6) CdTe QDs induced DNA damage. Olive tail moment and tail DNA content increased with exposure concentrations of CdTe QDs.
Then, interactions between CdTe QDs and proteins (serum albumin, catalase, Cu/Zn superoxide dismutases) were investigated using spectroscopy, isothermal titration calorimetry. The results show that the interactions between CdTe QDs and these three proteins are weak, and no profound conformational change of these proteins occurs. In addition, the analysis of catalase and Cu/Zn-SOD activity showed that CdTe QDs did not affect the function of these two antioxidant enzyme.
In this research, we explored the toxicity of CdTe QDs from the cellular and molecular perspectives, which provides valuable information to understand the toxicity of quantum dots in vitro and can be used to assist in the design of biocompatible and stable quantum dots.
首先,我们利用“一锅法”合成了谷胱甘肽包被的CdTe量子点,并对合成的CdTe量子点进行了表征。
随后,利用CCK-8法初步分析了CdTe量子点的细胞毒性,从整体上评价了CdTe量子点对小鼠肾小管上皮细胞及肝细胞活力的影响。在此基础上主要利用流式细胞术探讨了经量子点暴露后含量子点及镉离子的细胞比例与量子点细胞毒性的关系、细胞对CdTe量子点所引发的氧化应激反应(RC、MDA含量以及过氧化氢酶、SOD的活性指标)以及氧化应激失效导致的细胞凋亡。另外,通过单细胞凝胶电泳技术研究了CdTe量子点产生的基因毒性。得出的主要结论有：(1)CdTe量子点对这两种细胞均产生了细胞毒性。肝细胞对CdTe量子点的耐受性要高于肾小管上皮细胞。(2)CdTe量子点能够进入两种细胞内。经CdTe量子点染毒后仅少数细胞内含Cd2+。随着染毒浓度的增大,含量子点的细胞数目不断增加。倒置荧光显微镜下的观察也证实了量子点在细胞内的存在。(3)CdTe量子点在细胞内释放出镉离子。随着暴露浓度的增大,含Cd2+的细胞比例呈逐渐上升趋势。细胞内Cd2+浓度的上升与CCK-8测得的细胞活力下降呈现出一定的相关性。“木马”效应解释了细胞内存在镉离子的现象。(4)CdTe量子点诱发细胞产生氧化应激效应。细胞内ROS产生量随着CdTe量子点暴露浓度的增加而增加。对两种细胞内与氧化应激相关酶的研究发现SOD的活力上升,而过氧化氢酶的活力出现下降。(5)CdTe量子点诱导细胞凋亡。经CdTe量子点暴露后,小鼠肾小管上皮细胞及肝细胞中处于凋亡期的细胞占比出现上升。(6)CdTe量子点造成细胞DNA断裂损伤。Olive尾距及尾部DNA含量指标随着CdTe量子点暴露浓度的增加均呈现明显上升趋势。
之后,运用光谱学及等温滴定量热技术等方法分别研究了谷胱甘肽包被的CdTe量子点与血清白蛋白、过氧化氢酶以及Cu/Zn-SOD的相互作用。研究结果表明CdTe量子点与这三种蛋白的相互作用较弱,未改变蛋白的结构。疏水力是CdTe量子点与这三种蛋白相互作用过程中的主要驱动力。对过氧化氢酶以及SOD的活性分析显示CdTe量子点对这两种抗氧化酶的功能没有产生影响。
基于流式细胞术、光谱学及等温滴定量热等实验技术,本文从细胞及分子水平对水相合成的CdTe量子点的毒性进行了探讨,研究结果有助于设计及合成生物相容性及稳定性更佳的量子点,并为合理利用并最大限度地降低量子点的负面效应提供了理论参考。
Quantum dots are group IIB-VIA (eg, CdSe, CdTe) or group IIIA-VA (eg, InP, GaAs) semiconductor nanocrystals in the size range of2-100nm. Due to their symmetric and narrow emission spectrum, tunable fluorescence emission, broad excitation spectra and high resistance to photobleaching, quantum dots have superior optical properties over other organic fluorescent dyes, and present a broad potential for biomedical applications. With the development of quantum dots applications, chance of people exposed to such substances is growing. Numerous studies have shown that liver and kidney are the main target organs for cadmium-based quantum dots, which are also found for cadmium. Hepatocytes are widely used for evaluation of cytotoxicity of cadmium, and tubular epithelial cells constitute an established model in renal toxicology. Therefore, primary hepatocytes and renal tubular epithelial cells were used in this research as an evaluation model for cytotoxicity of CdTe QDs. In addition, nanomaterials may be covered with a 'corona' of biological molecules like proteins immediately upon contact with the physiological environment. This combination gives these nanoparticles new "biological identity" and subsequently determine the response of cells or organs, hence we also investigated the interactions of CdTe QDs and proteins, which related to the cytotoxicity of quantum dots. In this research, we explored the toxicity of CdTe QDs from the cellular and molecular perspectives. The major works and results are as follows:
Firstly, glutathione-capped CdTe QDs were synthesized via a one pot method, and the characterization of the CdTe QDs was presented.
The cytotoxicity of CdTe QDs were preliminary analysed by assessment of cell viability was performed using the Cell Counting Kit-8(CCK-8) assay. To investigate cytotoxicity mechanisms of CdTe QDs, the cells containing CdTe QDs and cadmium ion, ROS content, cell oxidative damage (MDA content, the activity of catalase and SOD), cell apoptosis were determined. Moreover, single cell gel electrophoresis assay was employed to study the genotoxicity of CdTe QDs. The experimental results showed that:(1) CdTe QDs causes cytotoxicity to these two types of cells. CdTe QDs induced more serious cytotoxicity injury on renal tubular epithelial cells than hepatocytes.(2) CdTe QDs can enter these two cells. Only a small number of cells were found to contain Cd2+. The number of cells containing CdTe QDs increased with the exposure concentration. The observation from inverted fluorescence microscope confirmed the presence of the QDs in the cells.(3) Cd2+was released from the CdTe QDs after internalization in cells. The number of cells containing Cd2+increased with the exposure concentration. Trojan horse-type mechanism explained the release of cadmium ions in the renal tubular epithelial cells and hepatocytes.(4) CdTe QDs induced oxidative damage to these two types of cells. Cellular ROS content increased with the exposure dose; MDA content and the activity of SOD increased synchronous with QDs concentrations, while the activity of catalase declined.(5) The CdTe QDs induced apoptosis in renal tubular epithelial cells and hepatocytes.(6) CdTe QDs induced DNA damage. Olive tail moment and tail DNA content increased with exposure concentrations of CdTe QDs.
Then, interactions between CdTe QDs and proteins (serum albumin, catalase, Cu/Zn superoxide dismutases) were investigated using spectroscopy, isothermal titration calorimetry. The results show that the interactions between CdTe QDs and these three proteins are weak, and no profound conformational change of these proteins occurs. In addition, the analysis of catalase and Cu/Zn-SOD activity showed that CdTe QDs did not affect the function of these two antioxidant enzyme.
In this research, we explored the toxicity of CdTe QDs from the cellular and molecular perspectives, which provides valuable information to understand the toxicity of quantum dots in vitro and can be used to assist in the design of biocompatible and stable quantum dots.
引文
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