Name: measurement of chitinase activity in biological samples
Uploaded: 2019-08-22T15:00:00
Description: Watch this Scientific Journal Video about Measurement of Chitinase Activity in Biological Samples at JoVE.com

这项研究得到了美国肺脏协会和美国胸腔学会对LS的奖项的支持。作者衷心感谢杰克·埃利亚斯博士和李春根博士提供转基因小鼠菌株。

所有动物程序都是根据耶鲁大学医学院的IACUC批准的规程进行的。
1. 鼠标样本收集
<ol><li>
麻醉
	<ol>
<li>使用氯胺酮和木兰素（100毫克/千克氯胺酮和10毫克/千克木兰辛）对小鼠进行麻醉。</li>
	</ol>
</li>
	<li>
血液样本采集
	<ol>
<li>通过对脚趾捏没有反应来确认麻醉的深度。</li>
		<li>手术打开胸腔以暴露心脏。</li>
		<li>将26.5G针头插入左心室，将血液收集...

<ol>
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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=Chitin+Recognition+via+Chitotriosidase+Promotes+Pathologic+Type-2+Helper+T+Cell+Responses+to+Cryptococcal+Infection.">Chitin Recognition via Chitotriosidase Promotes Pathologic Type-2 Helper T Cell Responses to Cryptococcal Infection.</a> PLOS Pathogens. 11 (3), e1004701 (2015).</li><li>Elmonem, M. A., vanden Heuvel, L. P., Levtchenko, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+Effects+of+Chitotriosidase+Enzyme.">Immunomodulatory Effects of Chitotriosidase Enzyme.</a> Enzyme Research. 2016, 1-9 (2016).</li><li>Ferrari, A. R., Gaber, Y., Fraaije, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+method+for+the+determination+of+glucose+in+blood.">Simple method for the determination of glucose in blood.</a> Proc Am Fed Clin Res. 2, 78 (1945).</li><li>Zarei, M., 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=Characterization+of+Chitinase+with+Antifungal+Activity+from+a+Native+Serratia+Marcescens+B4A.">Characterization of Chitinase with Antifungal Activity from a Native Serratia Marcescens B4A.</a> Brazilian Journal of Microbiology. 42 (297), 1017-1029 (2011).</li><li>Zhu, Z., 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=Acidic+Mammalian+Chitinase+in+Asthmatic+Th2+Inflammation+and+IL-13+Pathway+Activation.">Acidic Mammalian Chitinase in Asthmatic Th2 Inflammation and IL-13 Pathway Activation.</a> Science. 304 (5677), 1678-1682 (2004).</li><li>Hollak, C. E., van Weely, S., van Oers, M. H., Aerts, 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=Marked+elevation+of+plasma+chitotriosidase+activity.+A+novel+hallmark+of+Gaucher+disease.">Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease.</a> Journal of Clinical Investigation. 93 (3), 1288-1292 (1994).</li><li>Musumeci, M., Malaguarnera, L., Simpore, J., Barone, R., Whalen, M., Musumeci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitotriosidase+activity+in+colostrum+from+African+and+Caucasian+women.">Chitotriosidase activity in colostrum from African and Caucasian women.</a> Clinical Chemistry and Laboratory. 43 (2), 198-201 (2005).</li><li>Maddens, B., 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=Chitinase-like+Proteins+are+Candidate+Biomarkers+for+Sepsis-induced+Acute+Kidney+Injury.">Chitinase-like Proteins are Candidate Biomarkers for Sepsis-induced Acute Kidney Injury.</a> Mol Cell Proteomics. 11, 1-13 (2012).</li><li>Hector, 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=Chitinase+activation+in+patients+with+fungus-associated+cystic+fibrosis+lung+disease.">Chitinase activation in patients with fungus-associated cystic fibrosis lung disease.</a> Journal of Allergy and Clinical Immunology. 138 (4), 1183-1189 (2016).</li><li>Hall, A. J., Morroll, S., Tighe, P., G&#246;tz, F., Falcone, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chitotriosidase+is+expressed+in+the+eye+and+lacrimal+gland+and+has+an+antimicrobial+spectrum+different+from+lysozyme.">Human chitotriosidase is expressed in the eye and lacrimal gland and has an antimicrobial spectrum different from lysozyme.</a> Microbes and Infection. 10 (1), 69-78 (2008).</li><li>Kim, L. 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=AMCase+is+a+crucial+regulator+of+type+2+immune+responses+to+inhaled+house+dust+mites.">AMCase is a crucial regulator of type 2 immune responses to inhaled house dust mites.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (22), e2891-e2899 (2015).</li><li>Olkhovych, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitotriosidase+activity+as+additional+biomarker+in+the+diagnosis+of+lysosomal+storage+diseases.">Chitotriosidase activity as additional biomarker in the diagnosis of lysosomal storage diseases.</a> Ukrainian Biochemical Journal. 88 (1), 69-78 (2016).</li><li>Bouayadi, O., 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=Disease:+An+Underdiagnosed+Pathology+in+the+Eastern+Moroccan+Population.">Disease: An Underdiagnosed Pathology in the Eastern Moroccan Population.</a> eJIFCC. 30 (1), 82-87 (2019).</li><li>Tasci, 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=Efficacy+of+serum+chitotriosidase+activity+in+early+treatment+of+patients+with+active+tuberculosis+and+a+negative+sputum+smear.">Efficacy of serum chitotriosidase activity in early treatment of patients with active tuberculosis and a negative sputum smear.</a> Therapeutics and Clinical Risk Management. 8, 369-372 (2012).</li><li>Barone, R., Simpor&#233;, J., Malaguarnera, L., Pignatelli, S., Musumeci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+chitotriosidase+activity+in+acute+Plasmodium+falciparum+malaria.">Plasma chitotriosidase activity in acute Plasmodium falciparum malaria.</a> Clinica Chimica Acta. 331 (1-2), 79-85 (2003).</li><li>Kitamoto, S., 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=Chitinase+inhibition+promotes+atherosclerosis+in+hyperlipidemic+mice.">Chitinase inhibition promotes atherosclerosis in hyperlipidemic mice.</a> American Journal of Pathology. 183 (1), 313-325 (2013).</li><li>Kzhyshkowska, J., Gratchev, A., Goerdt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Chitinases+and+Chitinase-Like+Proteins+as+Indicators+for+Infl+Ammation+and+Cancer.">Human Chitinases and Chitinase-Like Proteins as Indicators for Infl Ammation and Cancer.</a> Biomark Insights. 2, 128-146 (2007).</li></ol>

赤子酶活性已成为预测疾病严重性、疾病进展、治疗效果和特定病原体存在的重要生物标志物18。虽然许多关于齐替酶作用的长期假设理论尚未经过实验证实，但新的研究为奇它酶和齐齐那酶等蛋白质在各种疾病中的作用提供了重要的见解2， 9，20.
此处描述的方案是测?...

奇青素是地球上仅次于纤维素的第二大多糖，是各种生物的主要结构成分，包括昆虫、真菌、酵母和藻类的外骨骼;一些脊椎动物也有奇丁1。奇西纳酶是一个酶家族，能够分解奇丁，并在从细菌到哺乳动物2、3等物种的进化过程中高度保存。除了奇他酶，哺乳动物也有丘蒂酸酶样的蛋白质，这些蛋白质在结合奇他素的能力上与奇它酶相似，但不同的是，它们缺乏酶能力来切切奇他4。
尽管奇他和奇它原体早已被研究——调查可追溯到20世纪初——但主要焦点一直是它们在昆虫和其他无脊椎动物中的作用。事实上，直到20世纪60年代，脊椎动物才被发现有丘台动物。使用基于甲苯的测定法，在包括蜥蜴和黑鸟在内的许多脊椎动物的消化道中发现了甲子酶，据推测，这种观察是由于食用了含甲氧基的有机体，如昆虫5.
哺乳动物有两种酶活性形式的甲酸酶：酸性哺乳动物甲壳酶（AMCase;也称为CHIT2和CHIA）和甲基锡酶（CHIT1）4。这两种蛋白质都能够水解奇他蛋白。然而，CHIT1在人类中更具有酶活性，几乎所有齐齐辛酶活性都来自CHIT1。4在小鼠中，Chit1和AMCase几乎同样地对整个齐齐辛酶活性6作出贡献。另一方面，齐齐那酶样蛋白质缺乏齐齐那酶活性。
Chit1主要通过巨噬细胞分泌，通常被认为是对含甲苯的病原体7的免疫反应。该酶也已被证明参与单核细胞成熟到M1和M2巨噬细胞亚型，即使没有基质甲肽8的存在。此外，它也可能涉及其他免疫细胞的成熟，包括T帮助器2型（Th2）细胞和嗜酸性粒细胞-如在隐球菌肺感染9中证明的情况。这些研究指出，奇塔因斯系统在免疫系统中有着复杂的作用。
近年来，Chit1水平被发现是40多种人类疾病的重要生物标志物，包括体球储存疾病、传染病、呼吸系统疾病、内分泌疾病、心血管疾病、神经系统疾病和其他疾病（第10页）。在许多这些疾病中，Chit1水平是疾病严重程度和治疗效果的有力预测。
由于奇质酶已经获得了作为包括高彻病等多种疾病的生物标志物的声誉，因此开发了工具和检测方法，以方便对奇他打酶的存在进行检测。较旧的方法包括沙勒斯的程序、根据血糖测试改编的方案以及 3，5 二硝基水杨酸 （DNS） 方法。然而，这些方法往往是时间敏感和技术上困难11。这两种测试的程序要求减少无机氧化剂，例如，在沙勒程序的情况下，铁氧体化物，产生一种颜色变化，只能分光光度测量。此外，这两个测试都涉及加热或沸腾步骤，既耗时又需要颜色开发12，13。
这里描述的是一种快速而简单的荧光测定，以确定哺乳动物样本14、15中的齐辛酶水平。此处使用的两个样本包括血清和支气管输液洗浴液 （BAL）;在母乳和尿液样本中也测量了齐辛酶活性，该技术可以在这些类型的样品以及任何其他生物液体16、17中进行。

<table>
	<tbody>
		<tr>
			<td>4-methylumbelliferone</td>
			<td>Sigma</td>
			<td>M1381</td>
			<td>Standard: commonly used in flourimetric assays for determination of enzyme activity</td>
		</tr>
		<tr>
			<td>4MU-GlcNAc2</td>
			<td>Sigma</td>
			<td>M9763</td>
			<td>fluorescent chitinase substrate for use in mouse samples</td>
		</tr>
		<tr>
			<td>4MU-GlcNAc3</td>
			<td>Sigma</td>
			<td>M5639</td>
			<td>fluorescent chitinase substrate for use in human samples</td>
		</tr>
		<tr>
			<td>Citric Acid-monohydrate</td>
			<td></td>
			<td></td>
			<td>for use in McIlvain Buffer</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td></td>
			<td></td>
			<td>for use in Stop Buffer at a concentration of 0.3 M</td>
		</tr>
		<tr>
			<td>Na2HPO4xH2O</td>
			<td></td>
			<td></td>
			<td>for use in McIlvain Buffer</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td></td>
			<td></td>
			<td>for use in Stop Buffer at a concentration of 12 g/L</td>
		</tr>
		<tr>
			<td>Vision Plate: Non-sterile, untreated black 96 well plate</td>
			<td>4titude</td>
			<td>4ti-0224</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of chitinase activity in biological samples

奇他酶是裂解奇他素的酶。即使在没有皮他当，哺乳动物体内（包括基线）中也有大量的皮塔那色。钱他酶的确切作用尚不清楚，但据信它在消化和宿主防御含奇他成的食物和病原体方面分别起着重要作用。最近的工作，包括我们的，已经表明了齐辛酶和齐齐酸酶样蛋白在宿主免疫和过敏性疾病中的重要作用。重要的是，Chitinase活动是疾病严重性的重要生物标志物，包括哮喘和肺纤维化等2型炎症性疾病。同样，像高彻病这样的遗传性疾病患者的齐齐纳酶水平显著升高，这不仅与疾病严重程度相关，而且作为治疗效果的可靠生物标志物。此处概述的协议描述了一种简单、快速和直接的方法，用于测量小鼠 BAL 或血清样本中的齐辛酶活性，并且由于酶的高度保守性，可以广泛地适应人类受试者和其他模型生物体。

此处显示的结果来自一项研究，该研究测量了野生型（C57BL/6）的血清和BAL样本中的奇汀酶活性，以及Chit1转基因小鼠（在C57BL/6背景上），该研究在多氧环素启动子上过度表达Chit1基因。我们的数据表明，血清和BAL样本在基线698.2~189.9 nmol/mL+h和485.7 = 114 nmol/mlμh时均具有可检测到的齐辛酶活性。在饮用水中使用多氧环素的Chit1基因过度表达4周，导致在BAL（4，575 ± 673.8 nmol/mL+h，p = 0.003）和血清1556 × 25...

Watch this Scientific Journal Video about Measurement of Chitinase Activity in Biological Samples at JoVE.com

Measurement of Chitinase Activity in Biological Samples

这里介绍的是一种测量脑油中齐辛酶活性的简单方法，如支气管活原或血清。

生物样品中赤子酶活性的测定

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...

这种方法允许一种更有效的方法来量化生物样品中的奇丁酶活性。奇丁酶活性已被证明在多种疾病中是诊断和治疗疗效的一个很好的标志。早期测量奇丁酶活性的方法往往耗时且难以执行，这两个问题都通过此处所示的测定解决了。这项技术将帮助实现潜在的奇蒂纳斯作为诊断和治疗标记，使其更容易测量奇丁酶活性;例如，它可以用来监测高彻病的治疗，因为奇丁酶活性已被证明在有效治疗期间减少。首先为测定准备基板、标准和解决方案。根据手稿方向准备 1x McIlvain 缓冲液，并用它来稀释甲酸素基材至 500 微摩尔。将标准 4-甲基苯甲酸酯溶解到止损缓冲液中浓度为 0.1 毫摩尔，以创建标准曲线库存溶液。对于每 10 个样品，将 2.17 毫升 1x McIlvain 缓冲液与 100 微升的奇汀基材混合，以创建工作溶液，然后将工作溶液的 95 微升移液器放入 96 井板的每个井中。将测试样品的五微升添加到每个井中，并将其混合到工作溶液中。盖上盘子并摇动5秒钟，然后在37摄氏度下孵育板15分钟，使酶反应发生。同时，稀释4-甲基甲基苯甲酸酯的库存溶液，形成5微摩尔工作标准溶液，根据手稿制备一系列稀释，形成标准曲线。从五微摩尔浓度开始，混合每个稀释井。孵育后，在每一个井中加入200微升停止缓冲液，以停止反应。将每个标准的 300 微升重复物添加到同一个板中，然后在 360 纳米的激发和 455 纳米的发射时读取板。该协议被成功地用于测量支气管和野生类型的血清流体和野生的血清液体和奇托特里奥西达酶过度表达转基因小鼠的奇丁酶活性。该检测证实，小鼠中CHIT-1基因的超量表达会导致奇丁酶活性增加。标准曲线用于根据样品的荧光确定奇丁酶。首次尝试协议时，要记住的最重要的事情是尽可能快速高效地添加样本，同时仍要彻底混合。首次开始时，由于潜伏期较短，每次运行样本数较少，以确保测量精度可能是有益的。

Research

JoVE Journal

Immunology and Infection

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

胞内钙的测量 2 +</sup在隔离的收缩淋巴管

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

科研

免疫与感染

Measurement of &#947;HV68 Infection in Mice

Measurement of ÃƒÅ½Ã‚Â³HV68 Infection in Mice

γHV68感染小鼠的测量

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Determining the Phagocytic Activity of Clinical Antibody Samples

确定临床抗体样品的吞噬活性

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

测量人体血浆中的凝血因子V活性，用酶联凝固分析

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1ÃƒÅ½Ã‚Â² in Human Monocyte-derived Dendritic Cells

激活和NLRP3炎性活性的人单核细胞来源的树突状细胞用IL-1β测量

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...