Name: subcellular fractionation from fresh and frozen gastrointestinal specimens
Uploaded: 2018-07-15T15:00:00
Description: Watch this Scientific Journal Video about Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens at JoVE.com

作者承认下洛佩兹和 Miren Telletxea 为视频录制和编辑提供的帮助。ACR 由 Ikerbasque 奖学金和协会 de Celiacos 马德里 (ACM) 的研究项目资助。JRB 由 ISCIII-PI16/00258 项目资助, 并由欧洲联盟 ERDF/英法基金会共同出资, "成为欧洲的一种方式"。是由巴斯克卫生部的一个研究项目赠款2015111068资助的。IRG 和 AJM 分别得到 UPV/EHU 和巴斯克教育部 predoctoral 奖学金资助。

这项研究获得了该大学医院伦理委员会的批准, 并在获得所有受试者或其父母的知情同意后进行分析。
1. 活检收集
注: 患者远端十二指肠活检标本可在常规诊断内窥镜下进行, 检查范围为2至10毫克。活检组织是由不同细胞类型组成的复杂组织, 包括上皮、免疫和内皮细胞。临床肠胃根据既定的临床指南执行内窥镜。
<ol><li>在内窥镜下, 将活检放在无菌滤纸...

<ol>
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Tissue handling, cryopreservation, extraction, and use for proteomic analysis.</a> Acta oncologica (Stockholm, Sweden). 45 (6), 643-661 (2006).</li><li>Hoffmann, K., 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=New+application+of+a+subcellular+fractionation+method+to+kidney+and+testis+for+the+determination+of+conjugated+linoleic+acid+in+selected+cell+organelles+of+healthy+and+cancerous+human+tissues.">New application of a subcellular fractionation method to kidney and testis for the determination of conjugated linoleic acid in selected cell organelles of healthy and cancerous human tissues.</a> Analytical and Bioanalytical Chemistry. 381 (6), 1138-1144 (2005).</li><li>Foster, L. J., 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=A+Mammalian+Organelle+Map+by+Protein+Correlation+Profiling.">A Mammalian Organelle Map by Protein Correlation Profiling.</a> Cell. 125 (1), 187-199 (2006).</li><li>Baghirova, S., Hughes, B. G., Hendzel, M. J., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+fractionation+and+isolation+of+subcellular+proteins+from+tissue+or+cultured+cells.">Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells.</a> MethodsX. 2, e440-e445 (2015).</li><li>B&#246;rner, A., 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=Subcellular+protein+extraction+from+human+pancreatic+cancer+tissues.">Subcellular protein extraction from human pancreatic cancer tissues.</a> BioTechniques. 46 (4), 297-304 (2009).</li><li>Antalis, T. M., Shea-Donohue, T., Vogel, S. N., Sears, C., Fasano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+disease:+protease+functions+in+intestinal+mucosal+pathobiology.">Mechanisms of disease: protease functions in intestinal mucosal pathobiology.</a> Nature clinical practice. Gastroenterology. 4 (7), 393-402 (2007).</li><li>Cutler, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein purification protocols. , (2004).</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bicinchoninic+Acid+(BCA)+Assay+for+Protein+Quantitation.">The Bicinchoninic Acid (BCA) Assay for Protein Quantitation.</a> The Protein Protocols Handbook. , 11-14 (1996).</li><li>McLane, L. 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这里描述的协议用于肠道活检的核和细胞质分馏。纯化的蛋白质不变性, 不仅可以用于西方印迹分析如图 2和3 , 但也在化验要求本机折叠的蛋白质, 如免疫沉淀, 电泳移动转移检测 (EMSA) 或本机聚丙烯酰胺凝胶电泳 (页)。
所述方法依赖于组织的机械均匀化和渗透裂解, 在不影响核膜的情况下爆裂细胞膜。其结果是将用于下游分析的细胞质和核?...

蛋白质参与细胞内几乎所有的生物功能, 其结构、数量或位置的任何变化都可能导致致病性的情况。组织样品分馏法是降低疾病相关蛋白质分析复杂性的有效方法。一些研究使用蛋白质定位信息或丰富一个特定细胞室的蛋白质, 因此, 一个完整的蛋白质可再生分馏的协议是有用的回答某些生物学问题。确定蛋白质的亚细胞定位和监测其在基础和疾病条件下的区划再分配或相互作用, 将有助于确定与疾病相关的功能差异1,2。因此, 该方法的目的是重现性分级小肠组织活检, 无论是新鲜和冷冻, 进入细胞质和核亚细胞室。
肠道活检标本通常获得在内窥镜程序3 (图 1), 可以有效地用于蛋白质定量或免疫沉淀研究。由于肠道上皮组织样本将含有可能直接参与疾病发病机制4的蛋白质, 肠道活检标本是成功鉴别肠道疾病特异性的一个非常有价值的来源。蛋白质。储存冷冻, 病人组织样本连同临床信息是蛋白质分析的有用资源, 一个简单和可重复的样品准备是提供细胞区划信息的关键问题, 使用限制数量的冷冻组织5。
有几个商业可用的工具包分离的细胞分数, 但这些是更昂贵的, 一般比这里提出的协议花费更多的时间。基于多级梯度离心和连续梯度的协议以前也被用于不同组织6、7中各种细胞间隔的分馏。但是, 保持连续渐变的一致性往往是一项艰巨的任务。先前已经描述了基于连续裂解膜的类似协议, 但通常需要两个以上的缓冲器, 而手的时间更长8 。
当分馏组织样品时, 请记住, 组织样本呈现细胞细胞和细胞基质相互作用, 它们既不存在于培养细胞中, 又在蛋白质提取过程中很重要。在不影响蛋白质质量的情况下, 细胞与胞外基质的适当分解将是肠道组织蛋白分离9的关键因素。在这个协议中, 细胞细胞和细胞矩阵的接触首先被打破, 释放细胞并允许缓冲区到达各个细胞。为了避免在分馏之前进行蛋白质间的混合, 必须在不改变细胞或核膜完整性的情况下进行接触击穿。
由于肠道黏膜10中各种蛋白酶的丰富, 在提取过程中控制蛋白质降解具有重要意义。为了尽量减少潜在的蛋白质降解, 必须将多种蛋白酶抑制剂包括在蛋白质提取缓冲器中。此外, 如果提取物将用于功能化验, 这是必要的, 以避免蛋白质变性或蛋白水解, 因为这将导致蛋白质活性的损失11。为此, 该协议的执行在4摄氏度, 新鲜的 phenylmethylsulfonyl 氟化物 (PMSF) 被添加到缓冲区在最后浓度为0.1 毫米之前, 使用进一步抑制蛋白质水解。
细胞分馏的第一步是组织破坏和细胞裂解。如前所述, 其目的是将细胞分解, 并以最小的损伤打开。肠道组织样品必须均匀化, 细胞裂解以达到最大的细胞膜断裂。在本协议中, 我们使用电动杵式搅拌机打破了在几秒钟内均匀化的肠道组织通过高速涡流行动。均匀化后, 组织细胞在低渗缓冲中孵化, 它会爆裂细胞膜, 但会使细胞核保持完好, 其次是添加非变性洗涤剂 (壬 phenoxypolyethoxylethanol) 和短涡来分离细胞核。从细胞质分数。胞浆切除后, 细胞细胞核膜爆裂成高渗缓冲液, 晃动。
本文提出的方法是在内镜手术中获得的新鲜和冰冻小肠活检的亚细胞分馏的适当方法, 在2至10毫克的重量范围内。该协议简单、重现性好, 可在一小时内使用基本的实验室设备和试剂进行。

<table border="0" cellpadding="0" cellspacing="0" style="width:657px;" width="658">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">HEPES</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">H4034-1kg</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">KCl</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P9333-1kg</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">EDTA</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">E9884-100G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">NaCl</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">5588886-1kg</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">DTT</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">10197777001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">PMSF</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">36978</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Proteinase and phosphatase inhibitors</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">A32959</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">NP-40&#160;</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">CA-630</td>
			<td>Detergent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">BCA assay</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">23227</td>
			<td>Protein quantification kit</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Disposable plastic pestles</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">Z359947-100EA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Dounce homogeneizer</td>
			<td style="width:128px;">VWR</td>
			<td style="width:119px;">431-0100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Microcentrifuge</td>
			<td style="width:128px;">Eppendorf</td>
			<td style="width:119px;">5415-R</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Shacker</td>
			<td style="width:128px;">IKA</td>
			<td style="width:119px;">MS3 basic</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcellular fractionation from fresh and frozen gastrointestinal specimens

本议定书的目的是分级人肠道组织获得的内窥镜进入核和细胞质室, 以定位分析特定的蛋白质或蛋白质复合体在不同的组织状态 (即,健康与疾病)。该方法对新鲜和冷冻小肠组织标本的分馏有重要意义;它是容易接近的所有实验室, 而不是费时。

图 1显示了一个具有完整绒毛和隐窝结构的小肠活检切片的苏木精的伊红染色。
使用本议定书的核和细胞质分馏的代表性结果见图 2和3。在图 2所示的实验中, 新的 (Fh) 和冰冻的 (组织) 活检是在这里提出的协议同时进行分割的, 并使用每个样本的20微克进行?...

Watch this Scientific Journal Video about Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens at JoVE.com

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

在这里, 我们提出了一个协议, 以执行一个简单的细胞分离的胞质和核蛋白在人类新鲜和冰冻肠道活检。

新鲜冷冻胃肠标本的亚细胞分离

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