Name: analysis of the ambient particulate matter-induced chromosomal aberrations using an in vitro system
Uploaded: 2016-12-21T15:00:00
Description: Watch this Scientific Journal Video about Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System at JoVE.com

这项工作是支持的，部分由生物学卓越研究[授权号1P20GM109005]，阿肯色空间格兰特财团通过美国国家航空和航天局[授权号NNX15AK32A]卫生中心的国家研究所和美国国家职业安全和健康研究所（NIOSH）授权号码2T420H008436。笔者想感谢克里斯托弗Fettes校对和编辑这篇稿子。

1.颗粒收集和准备<ol><li> 高容量总悬浮微粒采样器的校准 <ol><li>断开质量流量控制器的采样器马达和马达连接到一个稳定的交流电源（ 图1）。 注：在此过程中不使用的过滤器。 </li><li>校准器安装孔和顶部装载转接板到采样。拧紧顶部装载适配器压紧螺母牢固，以确保没有空气泄漏是否存在。允许采样马达升温到其正常的操作温度（约10-15分钟）。 </li><li>覆盖与手...

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Phys. 6 (3), 729-753 (2006).</li><li>P&#246;schl, U., 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=Atmospheric+aerosols:+composition,+transformation,+climate+and+health+effects.">Atmospheric aerosols: composition, transformation, climate and health effects.</a> Angew. Chem. Int. 44 (46), 7520-7540 (2005).</li><li>Hou, L., 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=Altered+methylation+in+tandem+repeat+element+and+elemental+component+levels+in+inhalable+air+particles.">Altered methylation in tandem repeat element and elemental component levels in inhalable air particles.</a> Environ. Mol. Mutagen. 55 (3), 256-265 (2014).</li><li>Miousse, I. R., 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=Epigenetic+alterations+induced+by+ambient+particulate+matter+in+mouse+macrophages.">Epigenetic alterations induced by ambient particulate matter in mouse macrophages.</a> Environ. Mol. Mutagen. 55 (5), 428-435 (2014).</li><li>Miousse, I. R., 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=In+Vitro+Toxicity+and+Epigenotoxicity+of+Different+Types+of+Ambient+Particulate+Matter.">In Vitro Toxicity and Epigenotoxicity of Different Types of Ambient Particulate Matter.</a> Toxicol. Sci. 148 (2), 473-487 (2015).</li><li>Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+instability+in+cancer+development.">Genomic instability in cancer development.</a> Genome Instability in Cancer Development. , 363-392 (2005).</li><li>Michael, S., Montag, M., Dott, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-inflammatory+effects+and+oxidative+stress+in+lung+macrophages+and+epithelial+cells+induced+by+ambient+particulate+matter.">Pro-inflammatory effects and oxidative stress in lung macrophages and epithelial cells induced by ambient particulate matter.</a> Environ Pollut. 183, 19-29 (2013).</li><li>Li, N., 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=Induction+of+heme+oxygenase-1+expression+in+macrophages+by+diesel+exhaust+particle+chemicals+and+quinones+via+the+antioxidant-responsive+element.">Induction of heme oxygenase-1 expression in macrophages by diesel exhaust particle chemicals and quinones via the antioxidant-responsive element.</a> J Immunol. 165 (6), 3393-3401 (2000).</li><li>Pathak, R., Ramakumar, A., Subramanian, U., Prasanna, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radio-sensitivities+of+human+chromosomes+1+and+2+in+one+donor+in+interphase-+and+metaphase-spreads+after+60Co+gamma-irradiation.">Differential radio-sensitivities of human chromosomes 1 and 2 in one donor in interphase- and metaphase-spreads after 60Co gamma-irradiation.</a> BMC Med. Phys. 9, 6649 (2009).</li><li>Pathak, R., Dey, S. K., Sarma, A., Khuda-Bukhsh, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxic+effects+in+M5+cells+and+Chinese+hamster+V79+cells+after+exposure+to+7+Li-beam+(LET=+60keV/&#181;m)+and+correlation+of+their+survival+dynamics+to+nuclear+damages+and+cell+death.">Genotoxic effects in M5 cells and Chinese hamster V79 cells after exposure to 7 Li-beam (LET= 60keV/&#181;m) and correlation of their survival dynamics to nuclear damages and cell death.</a> Mutat. Res. Genet. Toxicol. Environ. Mutagen. 628 (1), 56-66 (2007).</li><li>Pathak, R., Dey, S. K., Sarma, A., Khuda-Bukhsh, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+killing,+nuclear+damage+and+apoptosis+in+Chinese+hamster+V79+cells+after+irradiation+with+heavy-ion+beams+of+16+O,+12+C+and+7+Li.">Cell killing, nuclear damage and apoptosis in Chinese hamster V79 cells after irradiation with heavy-ion beams of 16 O, 12 C and 7 Li.</a> Mutat. Res. Genet. Toxicol. Environ. Mutagen. 632 (1), 58-68 (2007).</li><li>Chalbot, M. C., Kavouras, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+spectroscopy+for+determining+the+functional+content+of+organic+aerosols:+a+review.">Nuclear magnetic resonance spectroscopy for determining the functional content of organic aerosols: a review.</a> Environ. Pollut. 191, 232-249 (2014).</li></ol>

细胞遗传学研究或数字或染色体，主要是在中期扩展的结构，微观分析，为预测，风险评估和治疗各种疾病的关键信息。它现在公认该细胞遗传学异常与几种疾病，包括癌症的进展和发展相关。迄今为止，中国科学院已经在所有主要类型的肿瘤中发现。可诱导各种类型的细胞遗传学改变16的主要外部危险因素之一-的CA可以自发或通过外部或内部的刺激，如电离辐射（IR）的出现。 CA的辅助?...

据估计，暴露于颗粒物（PM）导致每年超过300万过量死亡，主要是从心肺疾病和肺癌1。的确，PM被确认为致癌由国际癌症研究机构（组1）人类，如肺癌的暴露的水平提高到下午的风险增加已经显示2。有趣的是，几乎所有的肿瘤细胞海港数值和/或结构染色体异常。颗粒有机碳是一种变型的，复杂的，和非均相混合物，其组成和大小分布取决于排放以及物理和化学变化。它占来自城市的PM2.5质量的35-55％（PM 2.5微米尺寸和更小）以及农村和大陆背景的PM 2.5质量3,4的60％以上。水溶性部分占有机气溶胶的30-90％。大量有机化合物已经确定，包括脂族烃，多环芳烃，和它们的氧化和硝化的衍生物，脂族醛和醇，游离脂肪酸和它们的盐，二羧酸，多官能的化合物，蛋白质和类腐殖质大分子（HULIS）使用色谱法加上质谱技术5-8。这些化合物代表微粒的有机碳小于10-20％，因此大多数的有机碳是未知9。 实验证据表明，细胞毒性，氧化应激和炎症中涉及的PM-有关的病理状态的发展。最近已显示，但是，暴露于PM也导致一些外遗传改变的，包括在重复元件的DNA甲基化的改变，在这两个实验在体外系统以及在人受试者10-12。特别感兴趣的是的效果PM卫星DNA - 主要和次要的卫星 - 这是在围绕染色体着丝粒的异色地区发现的。结果表明，这些效果可能是由自然持久的，因为他们可以在至少72小时曝光12后进行检测。改变DNA甲基化，特别是围绕着丝粒，可以在细胞分裂过程导致的卫星DNA的mRNA转录物，折衷染色体完整性累积，并随后导致的各种病理状态13的发展。 为CA的分析所造成的PM后生改建的终点细胞遗传学方法的适应性，因此具有很高的重要性。在这里，我们报告使用小鼠巨噬细胞RAW264.7模型系统中的CA 体外照射和分析环境PM收集和准备的办法。巨噬细胞包括防御的第一线对吸入异物，因此，叔他的细胞系用作建立和粒子毒理学11，12，14，15中的最常用的模型。

<table border="0" cellpadding="0" cellspacing="0" width="726">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Total suspended particulate sampler</td>
			<td style="width:179px;">Tisch Environmental</td>
			<td style="width:119px;">TE-5170</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">Bruker Avance III NMR spectrometer&#160;</td>
			<td style="width:179px;">Bruker</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TopSpin 3.5/pl2 software</td>
			<td style="width:179px;">Bruker</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">ACD/NMR Processor Academic Edition</td>
			<td style="width:179px;">ACD/Labs</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RAW264.7 murine macrophages</td>
			<td style="width:179px;">ATCC</td>
			<td style="width:119px;">ATCC TIB-71</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:261px;">High glucose DMEM GlutaMAX media</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10569010</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Fetal Bovine Serum</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">16000044</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">15140163</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">25200056</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">PBS, pH 7.4</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10010049</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:261px;">KaryoMAX Colcemid Solution in PBS</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">15212012</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">KaryoMAX Potassium Chloride Solution</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10575090</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Methanol (HPLC)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">A452N1-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Acetic Acid, Glacial</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP1185-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">Decon Contrad 70 Liquid Detergent</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">04-355-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Wright and Wright-Giemsa Stain Solutions</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">23-200733</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Permount Mounting Medium</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Axio Imager 2</td>
			<td style="width:179px;">Zeiss</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of the ambient particulate matter-induced chromosomal aberrations using an in vitro system

暴露于颗粒物质（PM）是一个主要的全球健康问题，这可能会损坏的各种细胞组分，包括核遗传物质。为了评估PM的核遗传完整性的影响，染色体结构畸变的得分在小鼠巨噬细胞RAW264.7细胞的中期扩展。 PM被从环境空气中具有高体积总悬浮颗粒采样收集。所收集的材料被溶解，并过滤以保持水溶性，细部。所述颗粒的特征在于用于通过核磁共振（NMR）谱的化学组成。不同浓度的颗粒悬浮液中加入到RAW264.7小鼠巨噬细胞的体外培养72小时的总暴露时间，与未处理的对照细胞一起。在曝光结束后，文化与秋水仙胺治疗逮捕细胞分裂中期。细胞，然后收获，用低渗溶液，定格在acetomethanol，D处理ropped到玻片上，最后用吉姆萨溶液染色。幻灯片进行检查，以评估使用亮场显微镜的结构染色体畸变（CA）的分裂中期利差在放大1000倍。 50至100中期扩展被打进每个治疗组。该技术适于检测结构染色体畸变（CA）的，如染色单体型断裂，染色单体型交流，偏心片段，双着丝粒和环染色体，双分钟，核内复制，和罗伯逊易位体外暴露于PM之后的。这是一个行之有效的细胞遗传学端点后天的改变相关联的有效方法。

极大，应注意在选择TSP采样器的位置，以及当进行收集一年中的时间。的化学组成，以及颗粒的尺寸可以基本上影响结果。收集的材料应该是对白色滤波器可见。普通鼠标中期扩展将有40偏心染色体。该技术的目的是要证明在异常染色体中处理的细胞与对照相比的比例的变化（或没有）。那么这种改变可以被量化（染色体异常的数量）和合格（类型异常）。 <p class="jove_...

Watch this Scientific Journal Video about Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System at JoVE.com

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

该协议与周围空气的微粒物质处理后描述了在RAW264.7小鼠巨噬细胞体外量化和染色体畸变的表征技术。

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Research

JoVE Journal

Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

In Vitro Transcription Assays and Their Application in Drug Discovery

In Vitro Transcription Assays and Their Application in Drug Discovery

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

科研

遗传学

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

使用旋转运动量化系统测量果蝇中的运动水平 (满足要求)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...