Name: toxicity study of zinc oxide nanoparticles in cell culture and in drosophila melanogaster
Uploaded: 2019-09-19T13:30:00
Description: Watch this Scientific Journal Video about Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster at JoVE.com

这项研究得到了补助金编号R706-000-043-490的支持。这项研究并不代表赠款发起人的观点。

1. 荧光活细胞分类 （FACS） 活细胞/固定细胞分析
<ol><li>声波 ZnO NP 在悬浮 15 分钟。</li>
	<li>使用1mg/mL ZnO NP库存溶液制备不同浓度的ZnO NP（例如0、10、25、50、100和200微克/mL），用于处理培养细胞。</li>
	<li>种子MRC5人类肺成纤维细胞（1 x 105细胞/孔）提前一天进入6孔培养板，然后用2 mL的ZnO NPs（三胞胎）处理细胞8小时、16小时和24小时。</li>
	<li>在每个时间点，通过300 x g离心收集细胞...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Drosophila+melanogaster.">The genome sequence of Drosophila melanogaster.</a> Science. 287, 2185-2195 (2000).</li><li>Ong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+nanoparticles+disrupt+germline+stem+cell+maintenance+in+the+Drosophila+testis.">Silver nanoparticles disrupt germline stem cell maintenance in the Drosophila testis.</a> Scientific Reports. 6, (2016).</li><li>Alaraby, M., Demir, E., Hernandez, A., Marcos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+potential+harmful+effects+of+CdSe+quantum+dots+by+using+Drosophila+melanogaster+as+in+vivo+model.">Assessing potential harmful effects of CdSe quantum dots by using Drosophila melanogaster as in vivo model.</a> Science of the Total Environment. 530-531, 66-75 (2015).</li><li>Barik, B. 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为了评估ZnO NP是否可诱导MRC5成纤维细胞凋亡，我们使用流式细胞测定法来区分细胞与坏死或凋亡细胞死亡。在正常活细胞中，磷脂酰丝氨酸（PS）在细胞膜处局部化。如果发生凋亡，PS被转移至血浆膜的细胞外传单，允许用荧光素标记的附件五（FITC附件五）29。另一方面，红荧光丙二蛋白（PI），一种核酸结合染料，对活细胞和凋亡细胞是无害的，但污渍死细胞30。<...

纳米技术是指纳米材料的应用，用于所有科学领域，包括医学、材料科学和生物化学。例如，以紫外线散射、化学传感、抗微生物特性以及高导电性著称的ZnO NPs被用于各种消费品的生产，如食品包装、化妆品、纺织品、橡胶、电池、汽车尾气处理催化剂以及生物医学相关应用1、2、3。
然而，基于ZnO NP的产品的迅速应用，导致人类接触ZnO NP的机会增加，这引起了人们对其对人类健康的潜在不利影响的关注。一些体外细胞研究表明，ZnO NPs可以诱导氧化应激、自噬相关细胞毒性、炎症和基因毒性4，5，6，7，8.值得注意的是，ZnO NPs的毒性被假定为由Zn溶解以释放Zn2+离子引起的，以及ZnO的表面反应性，导致细胞离子和代谢失衡，这些失衡与受损的离子平衡和抑制的子运输4，7，9，10。重要的是，研究表明，活性氧物种（ROS）的产生是ZnO NPs相关毒性的主要机制之一。ROS侮辱后抗氧化活性不足已被证明是导致细胞毒性和DNA损伤的原因9。在动物模型中也报告了ZnO NPs的毒性作用，包括啮齿动物1，斑马鱼11，12，以及无脊椎动物果蝇13。
果蝇是化学实体和纳米材料（NMs）14、15毒性筛选的成熟替代动物模型。重要的是，人类和果蝇之间有着高度的遗传和生理相似性，这证明使用果蝇作为体内模型来评估对环境污染物（如NMs）的生物反应。16.此外，由于果蝇体积小、寿命短、遗传性、易于操作、易于和具有成本效益的维护，使用果蝇有许多优点。此外，自2000年全基因组完全测序以来，果蝇被广泛用于遗传学、分子和发育生物学的研究，因此适合各种高通量筛选以及解决未解决的生物问题17，18，19，20，21。近年来，在果蝇中，有15、22、23、24号研究与使用不同类型核对素的免疫毒性有关。从使用果蝇的研究中获得的这些基本的新知识有助于深入了解我们对纳米毒理学的理解。
ROS是NPs引起的细胞毒性和基因毒性的众所周知的罪魁祸首，特别是基于金属的NPs25。ROS是含氧化学物种，具有比分子氧更高的活性。自由基，如超氧化物基（O2-），甚至非基分子，如过氧化氢（H2 O2）可以充当ROS。在正常生理条件下，它们需要维持细胞平衡26，然而，过量的ROS由于过量生产或抗氧化防御系统调节可能导致氧化应激，导致蛋白质损伤，脂质和脱氧核糖核酸（DNA）27。例如，随着ROS水平增加和谷胱甘肽（GSH）水平同时下降，三磷酸腺苷（ATP）合成发生中断，乳酸脱氢酶（LDH）水平增加，最终导致细胞死亡27。
在这里，我们提供使用培养的哺乳动物细胞和果蝇细胞进行细胞和遗传分析的协议，以确定ZnO NPs的潜在不利影响。图1概述了用于ZnO NPs毒性研究的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15% Methyl 4-Hydroxybenzoate</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cncCK6/TM3, Sb</td>
			<td></td>
			<td></td>
			<td>a gift from Dr. Kerppola T</td>
		</tr>
		<tr>
			<td>cornmeal, glucose, yeast brewer</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CyAn ADP with Summit Software</td>
			<td>DAKO</td>
			<td></td>
			<td>https://flow.usc.edu/files/2014/07/BC-Cyan-ADP-User-Guide-2016.pdf</td>
		</tr>
		<tr>
			<td>Dihydroethidium (Hydroethidine)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D11347</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Annexin V Apoptosis Detection Kit I</td>
			<td>BD biosciences</td>
			<td>556547</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucolin</td>
			<td>Supermarket</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRC5 human lung fibroblast</td>
			<td>ATCC</td>
			<td>CCL-171</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#8217;s Drosophila medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21720-024</td>
			<td></td>
		</tr>
		<tr>
			<td>vectashield antifade mounting medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>wild- type Canton-S; Sod2N308/CyO</td>
			<td>NIG-FLY</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Oxide Nanoparticles</td>
			<td>Sigma Aldrich</td>
			<td>721077</td>
			<td>Refer Sheet 2</td>
		</tr>
	</tbody>
</table>

toxicity study of zinc oxide nanoparticles in cell culture and in drosophila melanogaster

氧化锌纳米颗粒 （ZnO NPs） 的应用范围很广，但近年来有关 ZnO NP 相关毒性的报告数量迅速增加。然而，阐明ZnO NP诱导毒性的基本机制的研究很少。我们使用体外实验模型和体内实验模型确定了ZnO NPs的毒性曲线。在ZnO NP暴露的MRC5肺成纤维细胞中观察到细胞活力显著下降，表明ZnO Npps具有细胞毒性作用。同样，有趣的是，暴露于ZnO NPs的肠道在果蝇果蝇果蝇中表现出活性氧物种水平（ROS）的显著增加。需要进行更深入的研究，以便对消费者增加使用 ZnO NPs 进行风险评估。

NP暴露的细胞使用细胞染色试剂试剂盒进行处理，然后使用流式细胞测定法对细胞进行分类。ZnO NP处理的细胞（底部，右侧面板）表现出比对照细胞（R5，底部，左侧面板）更高的早期（R3）/晚期凋亡细胞（R6）的百分比。坏死细胞死亡用R4（顶部，右侧面板）表示（图2）。图2显示了FITC/附件五对ZnO NP处理的MRC-5成纤维细胞的测?...

Watch this Scientific Journal Video about Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster at JoVE.com

Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster

我们描述了一个详细的方案，用于评估氧化锌纳米粒子（ZnO NPs）的毒理学特征，特别是人类MRC5肺成纤维细胞中的细胞死亡类型和果蝇果蝇果蝇的ROS形成。

细胞培养和果蝇黑色素的氧化锌纳米颗粒的毒性研究

Zinc oxide nanoparticles (ZnO NPs) have a wide range of applications, but the number of reports on ZnO NP-associated toxicity has grown rapidly in recent ...

我们的方法概述了研究纳米毒性的简单而重复的方法，例如检查细胞死亡的类型和检查活性氧物种的产生。荧光激活细胞分拣是一种严格的格式，使我们能够研究细胞的亚细胞和生长不同类型的细胞死亡。DHE探针研究方法使我们能够检测和量化活性氧物种，然后进行流动细胞学、微观高含量成像和基于对对的荧光分析。首先，将含有氧化锌纳米颗粒的Eppendorf管放入氧化锌纳米颗粒溶液中，放入声波器中15分钟。使用每毫升一毫克的氧化锌纳米粒子储存各种浓度的氧化锌纳米粒子。然后，在生物安全柜中，将 MRC5 人类肺成纤维细胞种子到三个六孔培养板上，浓度为每孔10至第5个细胞的1倍。在37摄氏度下孵育细胞。一天后，用两毫升氧化锌纳米粒子处理细胞8小时、16小时或24小时。在每个时间点，将细胞收集到15毫升的离心管中，以300倍g的离心器进行5分钟。用磷酸盐缓冲的盐水清洗细胞颗粒两次，然后用1X结合缓冲液重新暂停细胞。接下来，在五微升碘化DNA染色中加入5个菲特基附件五染色的微升。在黑暗中的室温下孵育染色细胞15分钟。在通过流式细胞学对细胞进行排序之前，再用 400 微升 1X 结合缓冲液对样品进行补充。使用获得的中位数强度对条形图进行制表。现在使用移液器将不同浓度的一毫升纳米颗粒加入小瓶中，然后加入9毫升的飞食，使最终浓度为每毫升0.1毫克，每毫升0.25毫克，或每毫升氧化锌纳米颗粒0.5毫克。上下移液，彻底混合。允许含有氧化锌纳米颗粒的飞食在使用前至少冷却两到三个小时。然后麻醉成年的雄蝇和雌性苍蝇在小瓶与二氧化碳，并引入五公苍蝇和五只雌性苍蝇到每个小瓶五天。允许他们交配，并在食物表面产卵。麻醉后，取出父母的苍蝇，让卵子在室温下进一步发育，室温由四个不同的发育阶段组成。胚胎、幼虫、幼虫和成人阶段。72至120小时后，新鲜产卵发育成晚三星幼虫。使用钳子，从小瓶壁上收集幼虫进行分析。在具有解剖介质或 PBS 的解剖盘中，将幼虫放入溶液中。在立体显微镜下，用钳子的尖端做一个小孔，并打破幼虫的角质层。小心地拉出肠道。将肠道放入含有施耐德嗜血杆的1.5毫升微离心管中。加入一个微升的DHE探头，在室温下在黑暗中孵育10分钟。每五分钟用施耐德的中位清洗三次肠道。将肠子安装到玻璃幻灯片上，并提供 10 微升含有 DAPI 的防淡安装介质。在共和显微镜下捕捉图像。要测量荧光，请将使用荧光显微镜或共声激光扫描显微镜采集的荧光图像导入 ImageJ 软件。单击分析菜单，然后选择设置的测量值。选择输出度量，如区域综合强度和平均灰色值。选择无荧光区域以设置背景。单击度量值。将数据导出到电子表格中，并确定校正的总细胞荧光。构建条形图，并执行统计分析。在这项研究中，氧化锌纳米粒子处理细胞表现出比控制细胞R5更高的早期R3或晚期凋亡R6细胞的百分比。坏死细胞死亡用R4表示。从果蝇幼虫中提取的肠道被分为三个不同发育起源的离散域。也就是说， 前腿， 中， 和后。DHE 股票往往很快过期，因此请注意您使用的股票解决方案。或者，您可以尝试其他探测器，例如细胞罗氏，它旨在检测绿色信号中的细胞内 ROS，并且比 DHE 更可光。我们的协议为研究细胞内ROS生产提供了总体大纲，并为纳米毒性研究提供了ROS水平的量化。众所周知，DMSO 是 DHE 比 PBS 更好的溶剂。然而，DMSO是有毒的。因此，我想建议用户将学习的整个过程限制在30分钟以上。这是因为您可能会观察到误报结果，因为 DMSO 可能会导致细胞死亡。

Research

JoVE Journal

Immunology and Infection

Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

全身细菌感染与免疫防御的表型<em&gt;果蝇</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

枯否细胞分离的纳米粒子毒性测试

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...