纳米金棒对A549细胞毒性的机制研究
发布时间：2018-09-08 15:59
【摘要】：纳米金棒(Gold nanorods,AuNRs)是指尺度介于1-100nm之间的棒状纳米金颗粒,在光学特性、量子隧道效应、生物相容性、表面易修饰性和稳定性等方面具有独特的物理及化学属性,因此被认为是最具应用前景的纳米材料之一。近年来,随着纳米技术在生物医学领域的发展,AuNRs在细胞和动物活体成像、药物和基因传递以及多种疾病的诊断和治疗等方面的运用越来越多。然而,随着AuNRs广泛应用,人们接触AuNRs的机会日益增多,AuNRs暴露所引起的生物安全问题也受到越来越多的关注。因此,探究AuNRs对生物体可能潜在的影响及其机制是纳米金材料得到更加安全、广泛运用的基础。本研究拟首先采用不同浓度AuNRs处理A549细胞6h、12h和24h,然后通过CCK-8、LDH实验观察AuNRs对A549细胞活力、细胞膜损伤的影响。在此基础上,运用透射电镜观察AuNRs对细胞超微结构的影响,并初步判断AuNRs的细胞毒性。进一步,拟运用Western blot技术、激光扫描共聚焦显微技术和其他分子生物学方法来观察AuNRs细胞毒性是否与其通过损伤线粒体相关功能、促进氧化应激进而诱导细胞自噬有关,相关实验结果如下:一、AuNRs对A549细胞毒性的影响首先,将正常培养的A549细胞给予不同浓度AuNRs处理(0μg/ml,0.5μg/ml,1μg/ml,2μg/ml,4μg/ml),分别处理6h、12h、24h后观察其对细胞活力、细胞膜损伤和细胞超微结构的影响,实验结果如下:1.AuNRs对A549细胞活力的影响CCK-8实验结果表明,采用不同浓度AuNRs处理后,A549细胞活力呈剂量、时间依赖性降低。提示,AuNRs细胞毒性呈剂量、时间依赖性增加。2.AuNRs对A549细胞膜损伤的影响LDH实验结果表明,采用不同浓度AuNRs处理后,A549细胞呈现LDH渗漏且上清LDH呈剂量、时间依赖性增加。提示,AuNRs细胞毒性呈剂量、时间依赖性增加,AuNRs作用的浓度越高、时间越长,对细胞的毒性越大。3.AuNRs对A549细胞超微结构的影响透射电镜研究结果表明,AuNRs能够进入A549细胞,并以单个颗粒或聚集体的形式存在于细胞质和部分膜囊泡中,而细胞核未观察到AuNRs。此外,细胞中部分线粒体呈不同程度肿胀,并且自噬小体数目较对照组显著增加。二、aunrs对a549细胞自噬的影响研究发现,自噬是纳米材料细胞毒性的重要机制之一。那么aunrs对a549细胞毒性是否与自噬有关呢?我们进行以下研究,实验结果如下:1.aunrs对a549细胞自噬标志蛋白lc3的影响激光扫描共聚焦显微镜观察结果表明,不同浓度aunrs处理a549细胞6h后,lc3表达呈剂量依赖性增加。其中,con组lc3主要表达并集中于细胞核,而随着aunrs浓度的增加,lc3表达逐渐从细胞核转移到细胞质,细胞核lc3表达逐步减少并呈空泡化。再将a549细胞给予2μg/ml的aunrs分别处理6h、12h和24h并观察lc3表达情况,结果表明,a549细胞lc3表达呈时间依赖性增加。con组lc3主要表达并集中于细胞核,而随着aunrs暴露时间的增加,lc3表达逐渐从细胞核转移到细胞质,细胞核lc3表达逐步减少并呈空泡化。2.aunrs对a549细胞自噬相关蛋白的影响westernblot结果表明,不同浓度aunrs处理a549细胞6h后,lc3-ii、atg4、atg16和beclin1蛋白表达显着增加,而自噬底物蛋白p62表达水平显著降低。提示,aunrs诱导a549细胞发生自噬呈剂量、时间依赖性增加。3.cq能够抑制aunrs诱导的自噬westernblot结果表明,正常培养条件下con组atg16、lc3-ii少量表达,给予cq处理后atg16、lc3-ii表达较con组显著增加,表明cq可以抑制基础水平自噬溶酶体降解。给予aunrs处理后,atg16、lc-ii表达较对照组显著增加。aunrs和cq共同处理a549细胞6h后,atg16高表达可被cq逆转,而lc3-ii表达则进一步增加。这一结果表明,cq不仅可以抑制由aunrs诱导自噬的起始,而且可以抑制自噬溶酶体降解。三、氧化应激介导aunrs诱导a549细胞产生的自噬近年来有研究发现,氧化应激是诱导细胞自噬的重要原因。那么aunrs诱导a549细胞发生自噬是否与氧化应激有关?我们的实验结果如下:1.抗氧化剂mntbap和nac能够抑制aunrs诱导的自噬westernblot结果表明,con组中lc3-ii表达水平较低,mntbap、nac处理后其表达水平轻微降低但无统计学意义。而aunrs处理6h后,lc3-ii表达显著增加,并且这一效应能被mntbap、nac所逆转。提示,mntbap、nac可以改善aunrs诱导a549细胞产生的自噬。激光扫描共聚焦显微镜研究表明,con组、nac组中lc3表达较弱且主要集中于细胞核,表明其自噬水平很低。而aunrs处理6h后,lc3蛋白质从细胞核转移到细胞质,细胞核lc3表达逐步减少并呈空泡化。而抗氧化剂nac能够逆转上述效应。2.抗氧化剂MnTBAP和NAC能够逆转AuNRs诱导的ROS增加激光扫描共焦显微镜研究表明,不同浓度AuNRs处理A549细胞6h后,Con组未观察到ROS阳性细胞,表明其ROS水平很低。而随着AuNRs浓度的增加,阳性ROS细胞数量和绿色荧光亮度明显增加且呈剂量依赖性,表明AuNRs能够诱导ROS产生,浓度越高,ROS水平越高。而抗氧化剂Mn TBAP、NAC能够逆转上述效应。3.抗氧化剂MnTBAP和NAC能够改善AuNRs诱导细胞氧化应激状态T-AOC、GSH/GSSG检测结果表明,AuNRs处理组T-AOC、GSH/GSSG较Con组显著降低,表明AuNRs处理后细胞抗氧化能力显著降低,而MnTBAP、NAC处理6h后能够逆转上述效应。4.抗氧化剂NAC能够逆转AuNRs诱导线粒体功能的降低线粒体膜电位和ATP含量测定实验结果表明,AuNRs处理后其线粒体膜电位和ATP含量较Con组显著降低,表明AuNRs处理细胞后其线粒体功能降低,而NAC处理6h后能够逆转上述效应。Western blot结果表明,AuNRs处理组UCP2表达显著降低。NAC处理6h后UCP2蛋白表达显著增加,提示UCP2蛋白表达降低与线粒体功能改变有关。结论:1.AuNRs能够进入A549细胞,引起细胞超微结构改变。AuNRs具有一定的细胞毒性且呈剂量、时间依赖性增加,AuNRs浓度越高、作用时间越长,细胞毒性越大。2.AuNRs能够诱导A549细胞产生自噬并能被自噬抑制剂CQ所抑制,自噬标志蛋白LC3、自噬相关蛋白ATG16、ATG4、Beclin-1表达增加,而自噬底物蛋白P62表达降低且呈剂量依赖性。3.AuNRs能够影响A549细胞线粒体相关功能,进而导致细胞抗氧化应激能力降低、ROS水平增加,后者介导了AuNRs诱导A549细胞产生的自噬。
[Abstract]:Gold nanorods (AuNRs) are rod-like gold nanoparticles with a scale of 1-100 nm. They have unique physical and chemical properties in optical properties, quantum tunneling effect, biocompatibility, surface modification and stability, so they are considered as one of the most promising nanomaterials in recent years. With the development of biomedical technology, AuNRs are used more and more in cell and animal imaging, drug and gene delivery, diagnosis and treatment of various diseases. However, with the wide application of AuNRs, people have more and more opportunities to contact AuNRs, and the biological safety problems caused by AuNRs exposure have attracted more and more attention. Therefore, to explore the potential effect of AuNRs on organisms and its mechanism is the basis for the safety and widespread use of AuNRs. In this study, different concentrations of AuNRs were used to treat A549 cells for 6 h, 12 h and 24 h, and then CCK-8 and LDH were used to observe the effect of AuNRs on A549 cell viability and membrane damage. The effect of AuNRs on the ultrastructure of cells was observed by transmission electron microscopy, and the cytotoxicity of AuNRs was preliminarily judged. Furthermore, Western blot, laser scanning confocal microscopy and other molecular biology methods were used to observe whether the cytotoxicity of AuNRs was related to mitochondrial damage, promoting oxidative stress and inducing cells. The results were as follows: 1. The effects of AuNRs on cytotoxicity of A549 cells were studied. Firstly, the A549 cells were treated with different concentrations of AuNRs (0 ug/ml, 0.5 ug/ml, 1 ug/ml, 2 ug/ml, 4 ug/ml) for 6 hours, 12 hours and 24 hours respectively. The results were as follows: 1. The results of CCK-8 showed that the activity of A549 cells decreased in a dose-and time-dependent manner after treatment with different concentrations of AuNRs, suggesting that the cytotoxicity of AuNRs increased in a dose-and time-dependent manner. It was suggested that the cytotoxicity of AuNRs increased in a dose-and time-dependent manner. The higher the concentration of AuNRs, the longer the time, the greater the cytotoxicity. 3. The transmission electron microscopic study of the effect of AuNRs on the ultrastructure of A549 cells showed that AuNRs could enter A549 cells in a single particle. In addition, some mitochondria were swollen in different degrees and the number of autophagosomes was significantly increased compared with the control group. Second, the effect of AuNRs on autophagy of A549 cells was found to be one of the important mechanisms of cytotoxicity of nanomaterials. The results were as follows: 1. The effect of AuNRs on autophagy marker protein LC3 of A549 cells was observed by laser scanning confocal microscopy. The results showed that the expression of LC3 increased in a dose-dependent manner after AuNRs treatment for 6 hours. With the increase of AuNRs concentration, the expression of LC3 was gradually transferred from nucleus to cytoplasm, and the expression of LC3 in nucleus was gradually reduced and vacuolated. Then A549 cells were treated with 2 ug/ml AuNRs for 6 h, 12 h and 24 h respectively. The expression of LC3 in A549 cells increased in a time-dependent manner. The expression of LC3 gradually shifted from nucleus to cytoplasm with the increase of AuNRs exposure time. the expression of LC3 gradually decreased and vacuolated in nucleus. 2. the effect of AuNRs on autophagy-related proteins in A549 cells was studied by Western blot. the results showed that the expression of lc3-ii, atg4, atg16 and Beclin1 eggs in A549 cells treated with different concentrations of AuNRs for 6 hours. CQ could inhibit the expression of atg16 and LC3-II in con group. the expression of atg16 and LC3-II in con group was lower than that in con group. The expression of atg16 and lc-ii increased significantly after AuNRs treatment compared with the control group. After AuNRs and CQ treatment for 6 h, the high expression of atg16 could be reversed by cq, while the expression of LC3-II increased further. These results showed that CQ could not only inhibit the initiation of autophagy induced by aunrs. Oxidative stress mediated autophagy of A549 cells in recent years has found that oxidative stress is an important reason for inducing autophagy. then whether the autophagy of A549 cells induced by AuNRs is related to oxidative stress? Our experimental results are as follows: 1. antioxidant mntbap and NAC energy Inhibition of autophagy induced by AuNRs by Western blot showed that the expression of LC3-II was low in con group, but slightly decreased after treatment with mntbap and nac, but there was no significant difference. after treatment with AuNRs for 6 hours, the expression of LC3-II increased significantly, and this effect could be reversed by mntbap and nac. it suggested that mntbap and NAC could improve the production of A549 cells induced by aunrs. Confocal laser scanning microscopy showed that the expression of LC3 in con group and NAC group was weak and mainly concentrated in the nucleus, indicating that the level of autophagy was very low. Antioxidant MnTBAP and NAC could reverse the augmentation of ROS induced by AuNRs. Laser scanning confocal microscopy showed that after 6 hours of AuNRs treatment, no ROS-positive cells were observed in Con group, suggesting that the ROS level was very low. However, with the increase of AuNRs concentration, the number of ROS-positive cells and green fluorescence intensity increased significantly and was dose-dependent. Antioxidants MnTBAP and NAC could improve T-AOC of AuNRs-induced oxidative stress cells. GSH/GSSG test results showed that T-AOC and GSH/GSSG of AuNRs-treated cells were significantly lower than those of Con-treated cells. The results showed that the mitochondrial membrane potential and ATP content decreased significantly after AuNRs treatment, indicating that the mitochondrial mitochondrial membrane potential and ATP content decreased significantly after AuNRs treatment. The results of Western blot showed that the expression of UCP2 was significantly decreased in AuNRs treated group. The expression of UCP2 was significantly increased after 6 hours of NAC treatment, suggesting that the decrease of UCP2 protein expression was related to the change of mitochondrial function. Conclusion: 1. AuNRs could enter A549 cells and cause ultrastructural changes. The higher the concentration of AuNRs and the longer the duration of action, the greater the cytotoxicity. 2. AuNRs can induce autophagy in A549 cells and can be inhibited by autophagy inhibitor CQ. The expression of autophagy marker protein LC3, autophagy associated protein ATG16, ATG4 and Beclin-1 is increased, while the expression of autophagy substrate protein P62 is increased. AuNRs can affect the mitochondrial function of A549 cells in a dose-dependent manner, which leads to the decrease of antioxidant stress and the increase of ROS level, which mediates the autophagy of A549 cells induced by AuNRs.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08
本文编号：2230997
[Abstract]:Gold nanorods (AuNRs) are rod-like gold nanoparticles with a scale of 1-100 nm. They have unique physical and chemical properties in optical properties, quantum tunneling effect, biocompatibility, surface modification and stability, so they are considered as one of the most promising nanomaterials in recent years. With the development of biomedical technology, AuNRs are used more and more in cell and animal imaging, drug and gene delivery, diagnosis and treatment of various diseases. However, with the wide application of AuNRs, people have more and more opportunities to contact AuNRs, and the biological safety problems caused by AuNRs exposure have attracted more and more attention. Therefore, to explore the potential effect of AuNRs on organisms and its mechanism is the basis for the safety and widespread use of AuNRs. In this study, different concentrations of AuNRs were used to treat A549 cells for 6 h, 12 h and 24 h, and then CCK-8 and LDH were used to observe the effect of AuNRs on A549 cell viability and membrane damage. The effect of AuNRs on the ultrastructure of cells was observed by transmission electron microscopy, and the cytotoxicity of AuNRs was preliminarily judged. Furthermore, Western blot, laser scanning confocal microscopy and other molecular biology methods were used to observe whether the cytotoxicity of AuNRs was related to mitochondrial damage, promoting oxidative stress and inducing cells. The results were as follows: 1. The effects of AuNRs on cytotoxicity of A549 cells were studied. Firstly, the A549 cells were treated with different concentrations of AuNRs (0 ug/ml, 0.5 ug/ml, 1 ug/ml, 2 ug/ml, 4 ug/ml) for 6 hours, 12 hours and 24 hours respectively. The results were as follows: 1. The results of CCK-8 showed that the activity of A549 cells decreased in a dose-and time-dependent manner after treatment with different concentrations of AuNRs, suggesting that the cytotoxicity of AuNRs increased in a dose-and time-dependent manner. It was suggested that the cytotoxicity of AuNRs increased in a dose-and time-dependent manner. The higher the concentration of AuNRs, the longer the time, the greater the cytotoxicity. 3. The transmission electron microscopic study of the effect of AuNRs on the ultrastructure of A549 cells showed that AuNRs could enter A549 cells in a single particle. In addition, some mitochondria were swollen in different degrees and the number of autophagosomes was significantly increased compared with the control group. Second, the effect of AuNRs on autophagy of A549 cells was found to be one of the important mechanisms of cytotoxicity of nanomaterials. The results were as follows: 1. The effect of AuNRs on autophagy marker protein LC3 of A549 cells was observed by laser scanning confocal microscopy. The results showed that the expression of LC3 increased in a dose-dependent manner after AuNRs treatment for 6 hours. With the increase of AuNRs concentration, the expression of LC3 was gradually transferred from nucleus to cytoplasm, and the expression of LC3 in nucleus was gradually reduced and vacuolated. Then A549 cells were treated with 2 ug/ml AuNRs for 6 h, 12 h and 24 h respectively. The expression of LC3 in A549 cells increased in a time-dependent manner. The expression of LC3 gradually shifted from nucleus to cytoplasm with the increase of AuNRs exposure time. the expression of LC3 gradually decreased and vacuolated in nucleus. 2. the effect of AuNRs on autophagy-related proteins in A549 cells was studied by Western blot. the results showed that the expression of lc3-ii, atg4, atg16 and Beclin1 eggs in A549 cells treated with different concentrations of AuNRs for 6 hours. CQ could inhibit the expression of atg16 and LC3-II in con group. the expression of atg16 and LC3-II in con group was lower than that in con group. The expression of atg16 and lc-ii increased significantly after AuNRs treatment compared with the control group. After AuNRs and CQ treatment for 6 h, the high expression of atg16 could be reversed by cq, while the expression of LC3-II increased further. These results showed that CQ could not only inhibit the initiation of autophagy induced by aunrs. Oxidative stress mediated autophagy of A549 cells in recent years has found that oxidative stress is an important reason for inducing autophagy. then whether the autophagy of A549 cells induced by AuNRs is related to oxidative stress? Our experimental results are as follows: 1. antioxidant mntbap and NAC energy Inhibition of autophagy induced by AuNRs by Western blot showed that the expression of LC3-II was low in con group, but slightly decreased after treatment with mntbap and nac, but there was no significant difference. after treatment with AuNRs for 6 hours, the expression of LC3-II increased significantly, and this effect could be reversed by mntbap and nac. it suggested that mntbap and NAC could improve the production of A549 cells induced by aunrs. Confocal laser scanning microscopy showed that the expression of LC3 in con group and NAC group was weak and mainly concentrated in the nucleus, indicating that the level of autophagy was very low. Antioxidant MnTBAP and NAC could reverse the augmentation of ROS induced by AuNRs. Laser scanning confocal microscopy showed that after 6 hours of AuNRs treatment, no ROS-positive cells were observed in Con group, suggesting that the ROS level was very low. However, with the increase of AuNRs concentration, the number of ROS-positive cells and green fluorescence intensity increased significantly and was dose-dependent. Antioxidants MnTBAP and NAC could improve T-AOC of AuNRs-induced oxidative stress cells. GSH/GSSG test results showed that T-AOC and GSH/GSSG of AuNRs-treated cells were significantly lower than those of Con-treated cells. The results showed that the mitochondrial membrane potential and ATP content decreased significantly after AuNRs treatment, indicating that the mitochondrial mitochondrial membrane potential and ATP content decreased significantly after AuNRs treatment. The results of Western blot showed that the expression of UCP2 was significantly decreased in AuNRs treated group. The expression of UCP2 was significantly increased after 6 hours of NAC treatment, suggesting that the decrease of UCP2 protein expression was related to the change of mitochondrial function. Conclusion: 1. AuNRs could enter A549 cells and cause ultrastructural changes. The higher the concentration of AuNRs and the longer the duration of action, the greater the cytotoxicity. 2. AuNRs can induce autophagy in A549 cells and can be inhibited by autophagy inhibitor CQ. The expression of autophagy marker protein LC3, autophagy associated protein ATG16, ATG4 and Beclin-1 is increased, while the expression of autophagy substrate protein P62 is increased. AuNRs can affect the mitochondrial function of A549 cells in a dose-dependent manner, which leads to the decrease of antioxidant stress and the increase of ROS level, which mediates the autophagy of A549 cells induced by AuNRs.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08
【参考文献】
相关期刊论文 前2条
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2 陈波;陆燕蓉;陈又南;康裕建;程惊秋;;血管内皮衰老和氧化应激[J];生理科学进展;2015年01期
，本文编号：2230997
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