纳米硫化镉对人肾小管上皮细胞体外毒性研究
发布时间：2019-02-26 18:56
【摘要】：目的本研究通过体外细胞毒理学实验研究纳米硫化镉(nCdS)对人肾小管上皮细胞毒性作用,细胞选择人肾小管上皮细胞(HKC),探讨纳米态硫化镉对人肾小管上皮细胞(HKC)的氧化损伤过程及其相关机制。方法1)纳米硫化镉(nCds)实验材料的表征:分别使用透射电子显微镜(TEM)微结构分析和X-射线衍射探伤检测法(XRD)对实验所用纳米粒子进行表征验证,达到6~8nm尺度即可用于本研究。2)人肾小管上皮细胞(HKC)的传代培养:将人肾小管上皮细胞(HKC)进行体外传代培养并对其进行定性鉴定。3)生长情况影响:分别给予HKC不同剂量的纳米硫化镉(nCds),染毒后培养24h,同时设阴性对照组,采用噻唑蓝MTT法(不同剂量组)检测纳米硫化镉(nCds)对细胞的毒性增值影响。4)细胞形态观察:给予HKC不同剂量的纳米硫化镉(nCds)作用24h,同时设阴性对照组,采用AO/EB染色和DAPI染色后,放置在荧光显微镜下观察HKC形态的变化,透射电镜观察核内变化。5)实验研究纳米硫化镉对HKC内氧化应激的影响:分别检测纳米硫化镉处理后的细胞中ROS和GSH含量。6)探讨氧化应激机制:采用免疫组化法观察纳米硫化镉对细胞染毒处理后细胞内氧化代谢产物丙二醛(MDA)和抗氧化酶包括氧自由基(ROS)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物还原酶(GSH-Px)、细胞内谷胱甘肽(GSH)等物质含量变化情况。7)对人肾小管上皮细胞(HKC)内炎症反应机制探讨:应用蛋白免疫印迹法(western blotting法)检测纳米硫化镉(nCds)染毒处理后的细胞中核转录因子(NF-κB)蛋白表达水平,间接显示炎症反应的影响。结果本研究结果显示,随着实验组给予人肾小管上皮细胞(HKC)的纳米硫化镉(nCds)染毒处理剂量不断的增大,细胞存活率呈逐渐降低,与染毒剂量成反比;而生成促氧化物质ROS、MDA量升高,抗氧化物质GSH-Px、GSH生成量下降,同时观察到细胞内外的Cd2+浓度增加。实验中将染毒剂量划分为10.00、20.00、40.00、80.00μg/m L剂量组,实验随机分组染毒处理组的细胞存活率随染毒剂量的增加而下降,其变化具有统计学意义(P0.05)。结果显示各个剂量处理组的ROS和MDA含量以及40.00μg/mL剂量组的GSH与对照组相比较存在差别(P0.05)。随着纳米硫化镉(nCds)染毒剂量的增加,SOD、ROS、GSH-Px、GSH,40.00μg/m L剂量组的SOD、GSH-Px、GSH的含量下降,ROS、MDA的含量升高,且变化具有统计学意义(P0.05)。纳米硫化镉(nCdS)对HKC细胞中NF-κB蛋白表达水平的影响,除10μg/m L剂量组未见显著性差异,其他各个处理组均明与对照组存在差别(P0.05),细胞内核转录因子NF-κB蛋白表达水平随处理剂量的增加而逐渐升高。结论1.本研究验证了纳米硫化镉(nCds)毒性是通过增加活性氧和抑制抗氧化物质而诱发了氧化应激反应,引起线粒体的功能障碍,从而导致不同组织基因表达的改变。2.本研究验证了纳米硫化镉(nCds)毒性炎症反应是通过核转录因子NF-κB蛋白等相关蛋白发生作用的。3.探讨了纳米硫化镉(nCds)毒性机制中氧化应激、炎症反应和细胞凋亡相关路径的机制。4.纳米硫化镉(nCds)可以引起人肾小管上皮细胞(HKC)的毒性,其机制可能是氧化损伤和Cd2+释放的共同作用。
[Abstract]:Objective To study the toxicity of nano-sulfide (nCdS) to human renal tubular epithelial cells and to select the human renal tubular epithelial cells (HKC) by in vitro cytotoxicity. The oxidative damage of human renal tubular epithelial cells (HKC) and its related mechanisms were discussed. method 1) the characterization of nano-sulfur (nds) experimental materials: the nano-particles used in the experiment were characterized and verified by using a transmission electron microscope (tem) microstructure analysis and an x-ray diffraction detection method (xrd), respectively, 6-8nm scale was used in this study. 2) Subculture of human renal tubular epithelial cells (HKC): human renal tubular epithelial cells (HKC) were subcultured in vitro and their qualitative identification was carried out. 3) The effect of growth: nano-iron sulfide (nCds) with different doses of HKC, respectively, was given. The effect of nano-iron sulfide (nCds) on the cytotoxicity of the cells was detected by the method of MTT (different dose group) in the negative control group, and the effect of nano-iron sulfide (nCds) on the cytotoxicity of the cells was detected by the method of MTT (different dose group). The changes of the morphology of the HKC were observed with AO/ EB staining and DAPI, and the changes of the morphology of the HKC were observed under the microscope. The content of ROS and GSH in the cells treated with nano-iron sulfide was detected respectively. 6) The mechanism of oxidative stress was discussed: the malondialdehyde (MDA) and the antioxidant enzymes of the oxidized metabolites in the cells after the exposure to the cells were observed by the immunohistochemical method, including the oxygen free radicals (ROS). Changes of the content of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and intracellular glutathione (GSH) in human renal tubular epithelial cells (HKC) were discussed. The expression level of nuclear transcription factor (NF-EMAB) in the cells treated with nano-iron sulfide (nCds) was detected by Western blotting, and the effect of inflammatory reaction was indirectly shown. Results The results of the study showed that, with the increase of the dose of nano-iron sulfide (nCds) administered to human renal tubular epithelial cells (HKC) in the experimental group, the survival rate of the cells decreased gradually, which was inversely proportional to the exposure dose, and the amount of ROS and MDA was increased. The GSH-Px and GSH content of the anti-oxidant substances decreased, and the Cd2 + concentration inside and outside the cells was observed to increase. In the experiment, the dose group was divided into 10. 00, 20. 00, 40. 00, 80. 000.mu. g/ m L dose group, and the cell survival rate of the experimental group was decreased with the increase of the exposure dose, and the change was statistically significant (P0.05). The results showed that the content of ROS and MDA in each dose treatment group and the content of GSH in the 40. 000.mu. g/ mL dose group were different from the control group (P0.05). The contents of SOD, ROS, GSH-Px, GSH, GSH-Px and GSH decreased, and the content of SOD, GSH-Px and GSH decreased, and the content of ROS and MDA increased with the increase of the dose of nano-iron sulfide (nCds), and the content of ROS and MDA increased, and the change was of statistical significance (P0.05). The effect of nano-sulfide (nCdS) on the expression level of NF-EMAB protein in HKC cells was not found in the 10 & mu; g/ m L dose group, and there was a difference between the other treatment groups and the control group (P0.05). The expression of NF-EMAB protein in the cell core gradually increased with the increase of the treatment dose. Conclusion 1. The present study demonstrated that the toxicity of nano-sulfur (nds) was induced by the increase of active oxygen and the inhibition of antioxidant substances, which led to the dysfunction of the mitochondria, resulting in a change in the expression of different tissue genes. The present study demonstrated that the reaction of nano-sulfur (nCds) toxic inflammation is a function of the related proteins such as the nuclear transcription factor NF-EMAB protein. The mechanisms of oxidative stress, inflammatory response and apoptosis-related pathways in the nCds toxicity mechanism were discussed. Nano-iron sulfide (nCds) can lead to the toxicity of human renal tubular epithelial cells (HKC), and the mechanism may be the co-action of oxidative damage and Cd2 + release.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
本文编号：2431052
[Abstract]:Objective To study the toxicity of nano-sulfide (nCdS) to human renal tubular epithelial cells and to select the human renal tubular epithelial cells (HKC) by in vitro cytotoxicity. The oxidative damage of human renal tubular epithelial cells (HKC) and its related mechanisms were discussed. method 1) the characterization of nano-sulfur (nds) experimental materials: the nano-particles used in the experiment were characterized and verified by using a transmission electron microscope (tem) microstructure analysis and an x-ray diffraction detection method (xrd), respectively, 6-8nm scale was used in this study. 2) Subculture of human renal tubular epithelial cells (HKC): human renal tubular epithelial cells (HKC) were subcultured in vitro and their qualitative identification was carried out. 3) The effect of growth: nano-iron sulfide (nCds) with different doses of HKC, respectively, was given. The effect of nano-iron sulfide (nCds) on the cytotoxicity of the cells was detected by the method of MTT (different dose group) in the negative control group, and the effect of nano-iron sulfide (nCds) on the cytotoxicity of the cells was detected by the method of MTT (different dose group). The changes of the morphology of the HKC were observed with AO/ EB staining and DAPI, and the changes of the morphology of the HKC were observed under the microscope. The content of ROS and GSH in the cells treated with nano-iron sulfide was detected respectively. 6) The mechanism of oxidative stress was discussed: the malondialdehyde (MDA) and the antioxidant enzymes of the oxidized metabolites in the cells after the exposure to the cells were observed by the immunohistochemical method, including the oxygen free radicals (ROS). Changes of the content of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and intracellular glutathione (GSH) in human renal tubular epithelial cells (HKC) were discussed. The expression level of nuclear transcription factor (NF-EMAB) in the cells treated with nano-iron sulfide (nCds) was detected by Western blotting, and the effect of inflammatory reaction was indirectly shown. Results The results of the study showed that, with the increase of the dose of nano-iron sulfide (nCds) administered to human renal tubular epithelial cells (HKC) in the experimental group, the survival rate of the cells decreased gradually, which was inversely proportional to the exposure dose, and the amount of ROS and MDA was increased. The GSH-Px and GSH content of the anti-oxidant substances decreased, and the Cd2 + concentration inside and outside the cells was observed to increase. In the experiment, the dose group was divided into 10. 00, 20. 00, 40. 00, 80. 000.mu. g/ m L dose group, and the cell survival rate of the experimental group was decreased with the increase of the exposure dose, and the change was statistically significant (P0.05). The results showed that the content of ROS and MDA in each dose treatment group and the content of GSH in the 40. 000.mu. g/ mL dose group were different from the control group (P0.05). The contents of SOD, ROS, GSH-Px, GSH, GSH-Px and GSH decreased, and the content of SOD, GSH-Px and GSH decreased, and the content of ROS and MDA increased with the increase of the dose of nano-iron sulfide (nCds), and the content of ROS and MDA increased, and the change was of statistical significance (P0.05). The effect of nano-sulfide (nCdS) on the expression level of NF-EMAB protein in HKC cells was not found in the 10 & mu; g/ m L dose group, and there was a difference between the other treatment groups and the control group (P0.05). The expression of NF-EMAB protein in the cell core gradually increased with the increase of the treatment dose. Conclusion 1. The present study demonstrated that the toxicity of nano-sulfur (nds) was induced by the increase of active oxygen and the inhibition of antioxidant substances, which led to the dysfunction of the mitochondria, resulting in a change in the expression of different tissue genes. The present study demonstrated that the reaction of nano-sulfur (nCds) toxic inflammation is a function of the related proteins such as the nuclear transcription factor NF-EMAB protein. The mechanisms of oxidative stress, inflammatory response and apoptosis-related pathways in the nCds toxicity mechanism were discussed. Nano-iron sulfide (nCds) can lead to the toxicity of human renal tubular epithelial cells (HKC), and the mechanism may be the co-action of oxidative damage and Cd2 + release.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
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