Name: from a 2de-gel spot to protein function: lesson learned from hs1 in chronic lymphocytic leukemia
Uploaded: 2014-10-19T16:00:00
Description: Watch this Scientific Journal Video about From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia at JoVE.com

我们感谢淋巴组织的恶性肿瘤单位，Elisa的10克勤，肥姐Scarfò，马西莫·阿莱西奥，安东尼奥·孔蒂，安吉拉八尺，和Angela郭居静。 该项目支持：ASSOCIAZIONE ITALIANA每拉Ricerca SUL巨蟹座AIRC（调查员补助金和特别节目分子肿瘤学 - 5千分数＃9965） CS和BA设计的研究，进行了实验，分析数据和撰写文章。 MTSB，UR，FBPR协助撰写稿件。 PG和FCC的设计提供了研究患者的标本和临床资料。

注：所有组织样品与圣拉斐尔医院（米兰，意大利）的机构审查委员会批准获得。 1，人体组织样本和细胞纯化（图1） 注：淋巴细胞白血病从CLL患者的外周血（PB）根据Mulligan 等人获得，确诊9。 1.1）从PB白血病细胞分离<ol><li>与肝素钠真空采血管的血液收集管收集的PB。 </li><li>传输血液入15ml离心管，以50微升/毫升的全...

<ol>
	<li>Caligaris-Cappio, F., Ghia, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+in+chronic+lymphocytic+leukemia:+are+we+getting+closer+to+understanding+the+pathogenesis+of+the+disease.">Novel insights in chronic lymphocytic leukemia: are we getting closer to understanding the pathogenesis of the disease.</a> J Clin Oncol. 26, 4497-4503 (2008).</li><li>Rawstron, A. 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T., Scielzo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+microenvironment+wires+the+natural+history+of+chronic+lymphocytic+leukemia.">How the microenvironment wires the natural history of chronic lymphocytic leukemia.</a> Seminars in cancer biology. , (2013).</li><li>Damle, R. 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=Ig+V+gene+mutation+status+and+CD38+expression+as+novel+prognostic+indicators+in+chronic+lymphocytic+leukemia.">Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia.</a> Blood. 94, 1840-1847 (1999).</li><li>Lanham, 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=Differential+signaling+via+surface+IgM+is+associated+with+VH+gene+mutational+status+and+CD38+expression+in+chronic+lymphocytic+leukemia.">Differential signaling via surface IgM is associated with VH gene mutational status and CD38 expression in chronic lymphocytic leukemia.</a> Blood. 101, 1087-1093 (2003).</li><li>Wiestner, 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=ZAP-70+expression+identifies+a+chronic+lymphocytic+leukemia+subtype+with+unmutated+immunoglobulin+genes,+inferior+clinical+outcome,+and+distinct+gene+expression+profile.">ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile.</a> Blood. 101, 4944-4951 (2003).</li><li>Mulligan, C. 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<html>该原稿的目的在于描述一个优化的协议中涉及白血病的发病机制中的分子的识别，即使这种方法可以扩展到其它的疾病和/或其他类型的样品16-18。通过比较2亚CLL患者，良好与不良预后19的蛋白质组曲线，我们表明，它们在HS1的磷酸化状态的不同，它的激活影响白血病细胞的迁移和粘附能力。 相对于其他方法，在手稿，2DE凝胶和MS给出的蛋白质组技?...

慢性淋巴细胞白血病（CLL），在西方国家中最常见的成人白血病，来源于单克隆肿瘤CD5 + B淋巴细胞的积累和在临床上是非常不均匀的。它可以作为一个预白血病形式，定义为单克隆B细胞淋巴细胞增多（MBL）1,2,3。在其他情况下这种疾病可以出现任何与懒惰的临床过程，可以无需治疗前数年保持稳定，或作为一个渐进的chemorefractory疾病，预后极差，尽管治疗。最后，它可能会发展成一种经常致命的高恶性度淋巴瘤（里氏综合征-RS）2,3,4。患者通常被分为两个主要亚群：好的和坏的预后，根据一组预后因素，提供对疾病预后和生存预测因素的补充信息。临床异质性可能反映了生物异质性。许多基因，微环境和CEllular因素已被证明同意对疾病发病机理虽然没有统一的机制，已发现5。详细地说，一些研究已经表明，在疾病的临床过程的差别可以通过的具有预后价值6,7,8某些生物标记物的存在（或不存在）被部分地说明。这些数据表明，更好的了解该疾病的生物学可用于病理学的临床管理提供额外的提示，由两个预后标志物和治疗靶的鉴定。 本研究的目的是为了表明如何不同的蛋白质组技术的组合可用于参与CLL发作和进展的蛋白质的鉴定。我们的做法表明，它有可能联合起来基本蛋白质组学和临床资料5。 在本工作流程中，主从选择的患者的白血病细胞（良好与不良的预后）被分离和细胞裂解物，然后用于获得蛋白质的地图。 2DE允许的细胞群体，包括翻译后修饰的蛋白质分布图的特性，从而使每个蛋白的生物学活性的间接信息。全细胞裂解物通过基于等电点的第一维运行，随即通过上是基于其分子量解析蛋白质在聚丙烯酰胺凝胶上运行的第二尺寸。蛋白质组图谱进行比较分析允许差异表达的蛋白质的识别（无论是在数量和翻译后修饰而言）作为凝胶上的斑点可被切割并通过质谱法进行分析。每名候选人的角色可以由不同的试验来再利用。 这种方法中，只限于CLL在当前的手稿，可以很容易地扩展到其他疾病/样品，从而提供关于proteomi信息C /两组之间的生物学差异（ 即病理性与正常，刺激比未受刺激，野生型与击倒）。

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Name of Material/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Acetonitril</td>
			<td style="width:160px;">MERCK</td>
			<td style="width:111px;">61830025001730&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ammonium Bicarbonate</td>
			<td>SIGMA</td>
			<td style="width:111px;">A6141-500G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">1,4-Dithioerythritol</td>
			<td>SIGMA</td>
			<td style="width:111px;">D9680-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Iodoacetamide</td>
			<td>SIGMA</td>
			<td style="width:111px;">I6125-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Calcium chloride dihydrate</td>
			<td>SIGMA</td>
			<td style="width:111px;">223506-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Trypsin, Sequencing Grade</td>
			<td style="width:160px;">ROCHE</td>
			<td style="width:111px;">11418475001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-cyano-4-hydroxycinnamic acid</td>
			<td>SIGMA</td>
			<td style="width:111px;">C2020-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Trifluoroacetic acid</td>
			<td>SIGMA</td>
			<td style="width:111px;">T6580</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">ZipTip&#181;-C18</td>
			<td>MILLIPORE</td>
			<td>ZTC18M096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">RosetteSep Human B Cell Enrichment Cocktail</td>
			<td>STEMCELL</td>
			<td>15064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">PBS</td>
			<td>EUROCLONE</td>
			<td>ECB4004L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">FBS</td>
			<td>DOMINIQUE DUTSCHER</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Lymphoprep</td>
			<td>SENTINEL DIAGNOSTICS</td>
			<td>1114547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Trypan Blue</td>
			<td>SIGMA</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD19</td>
			<td>BECKMAN COULTER</td>
			<td>082A07770</td>
			<td>ECD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD5</td>
			<td>BECKMAN COULTER</td>
			<td>082A07754</td>
			<td>PC5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD3</td>
			<td>BECKMAN COULTER</td>
			<td>082A07746</td>
			<td>FITC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD14</td>
			<td>BD</td>
			<td>345785</td>
			<td>PE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD16</td>
			<td>BECKMAN COULTER</td>
			<td>082A07767</td>
			<td>PC5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD56</td>
			<td>BECKMAN COULTER</td>
			<td>082A07789</td>
			<td>PC5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ettan IPGphor 3 Isoelectric Focusing Unit</td>
			<td style="width:160px;">GE-Healthcare</td>
			<td>11-0033-64&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Urea</td>
			<td>SIGMA</td>
			<td>U6504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Tris-Hcl Buffer</td>
			<td>BIO-RAD</td>
			<td>161-0799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CHAPS</td>
			<td>SIGMA</td>
			<td>C2020-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DTT</td>
			<td>SIGMA</td>
			<td>D9163-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">IPGstrips</td>
			<td>GE-Healthcare</td>
			<td>17-1233-01&#160;</td>
			<td>Linear pH 4-7 18cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">IPGbuffer</td>
			<td>GE-Healthcare</td>
			<td>17-6000-86</td>
			<td>pH 4-7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Glycerol</td>
			<td>SIGMA</td>
			<td>G6279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">PROTEAN II XL Cell</td>
			<td>BIO-RAD</td>
			<td>165-3188</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">SDS</td>
			<td>INVITROGEN</td>
			<td>NP0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acrilamide</td>
			<td>BIO-RAD</td>
			<td>161-0156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Agarosio</td>
			<td>INVITROGEN</td>
			<td>16500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Methanol</td>
			<td>SIGMA</td>
			<td>32213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acetic Acid</td>
			<td>VWR</td>
			<td>631000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Formalin</td>
			<td>BIO-OPTICAL</td>
			<td>05-K01009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ethanol</td>
			<td>VWR</td>
			<td>9832500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium Thiosulfate</td>
			<td>FLUKA</td>
			<td>72049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Silver Nitrate</td>
			<td>FLUKA</td>
			<td>85228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular Dynamics Personal SI Laser Densitometer</td>
			<td>Amersham Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageMaster 2D Platinum 5.0</td>
			<td>Amersham Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium Carbonate</td>
			<td>MERCK</td>
			<td>A0250292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">MALDI-TOF Voyager-DE STR</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Data Explorer software (version 3.2)&#160;</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">transwell chambre 6.6 mm diameter</td>
			<td>CORNING</td>
			<td>3421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">RPMI</td>
			<td>EUROCLONE</td>
			<td>ECB2000L</td>
			<td>WITH L-GLUTAMINE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Gentamicin</td>
			<td>SIGMA</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">SDF-1</td>
			<td>PREPROTECH</td>
			<td>300-28A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Flat-bottom 96-well plate</td>
			<td>BECTON DICKINSON</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">BSA</td>
			<td>SANTA CRUZ</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">ICAM-1</td>
			<td>R&#38;D Systems</td>
			<td>720-IC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CMFDA</td>
			<td>Life Thechnology</td>
			<td>C7025</td>
			<td>Green</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">IgM</td>
			<td>SOUTHERNBIOTECH</td>
			<td>2020-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Paraformaldehyde</td>
			<td>SIGMA</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Saponine</td>
			<td>SIGMA</td>
			<td>S-7900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Phalloidin&#160;</td>
			<td>Life Thechnology</td>
			<td>A12379</td>
			<td>Alexa fluor 488</td>
		</tr>
	</tbody>
</table>

from a 2de-gel spot to protein function: lesson learned from hs1 in chronic lymphocytic leukemia

参与肿瘤发生和发展的分子的鉴定是基本的理解疾病的生物学和，其结果是，对于患者的临床管理。在目前的工作中，我们将介绍对参与慢性淋巴细胞白血病（CLL）的进展分子识别的优化蛋白质组学的方法。具体而言，白血病细胞裂解液是由二维电泳（2DE）解决，如可视化的双向凝胶电泳凝胶“点”。蛋白质组图谱进行比较分析允许差异表达的蛋白质的鉴定（在数量和翻译后修饰而言），该被拾取，分离并鉴定的质谱（MS）。所识别的候选者的生物学功能可以通过不同的测定（ 即迁移，粘附和F-肌动蛋白聚合）进行测试，我们已经对原代白血病细胞中进行优化。

我们分离的原代白血病B细胞形成CLL患者的PB和我们分析蛋白质组学地图。样品（n = 104），根据每个患者的（坏的预后与预后良好），并进行双向凝胶电泳凝胶的对比分析，临床特征主要在两个子集进行分组。 允许的分析鉴定斑两个子集之间的差异表达的（在存在/不存在或移凝胶上的术语，意味着改变翻译后修饰; N≈16）。我们切下选定斑点从2DE凝胶和被还原和烷基化?...

Watch this Scientific Journal Video about From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia at JoVE.com

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

在这里，我们描述了一种协议，它把两个蛋白质组学技术，即2维电泳（2DE）和质谱（MS）来识别差异表达/翻译后修饰的蛋白质中的两个或多个基团主样品。这种做法，再加上功能性实验，允许预后标志物/治疗靶点的鉴定和表征。

从2DE凝胶点在蛋白质功能：在课慢性淋巴细胞白血病学到HS1

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Research

JoVE Journal

Medicine

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

Technique to Collect Fungiform Taste Papillae from Human Tongue

技术收集人舌菌状（味道）乳头

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

科研

医学

Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia

利用重复经颅磁刺激改善脑卒中患者的语言功能与慢性非流利性失语

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this ...

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

干生生物标志物的血斑收藏最大限度地参与人口研究

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

膀胱胀诱发内脏运动反应为膀胱疼痛小鼠模型

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

血管功能在慢性肾脏疾病的评估

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型：教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

慢性血栓栓塞性肺动脉高压和右心室功能的仔猪评估

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Modeling Chemotherapy Resistant Leukemia In Vitro

Modeling Chemotherapy Resistant Leukemia In Vitro

建模化疗耐药白血病<em&gt;体外</em

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

A Protocol to Acquire the Degenerative Tenocyte from Humans

从人类获得变性 Tenocyte 的一种协议

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical ...

Protocol to Create Chronic Wounds in Diabetic Mice

在糖尿病小鼠中创建慢性伤口的协议

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...