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/毫升的全血添加人B细胞富集鸡尾酒。搅拌均匀，保温20分钟，在室温下。 </li><li>稀释样品用PBS +2％FBS等体积，轻轻混匀，小心叠加血在聚蔗糖，确保不突破的接口。使用至少1份聚蔗糖的2份稀释样品（ 即 4毫升聚蔗糖+10毫升血液在15ml吨UBE）。 </li><li>离心机在400 XG，在室温（20℃）20分钟，用不加制动。单核细胞会在界面处。 </li><li>小心吸出以回收细胞并将其放置到一个新的管的接口。一旦用PBS离心300 XG在黑暗中5分钟，洗涤细胞，4℃。沉淀会在底部。 </li><li>弃上清。通过轻弹管来回用手指悬浮在剩余的上清液中沉淀。稀释，用10ml的完全RPMI培养基中的小球（RPMI 1640补充有10％胎牛血清和15毫克/毫升庆大霉素）和计数用台盼蓝排除法的细胞。 </li></ol> 1.2）纯度评价注：所有制剂纯度必须始终在99％以上。 <ol><li>孵育100微升纯化的细胞悬浮液与下列抗体（市售;见表材料）：CD3 FITC（5微升/ TES吨），CD14 PE（5微升/试验），CD19 ECD（6微升/试验），CD16-CD56 PC5（2微升/试验），CD5 PC7在（4微升/试验），进行20分钟，在4℃下流式细胞仪管。 </li><li>洗用4ml的PBS（离心5分钟，在300 XG）的样品，并检查其纯度通过流式细胞cytometry.Check在剧情CLL细胞（&gt; 99％），其共表达CD19和CD5在其表面上作为％在图1中示出（6）。确保准备工作几乎毫无NK细胞，T淋巴细胞和单核细胞在情节，这应该是接近零检测CD3 +，CD14和CD16-56的％。 </li></ol> 1.3）样品储存<ol><li>用于蛋白组学研究中，收集25×10 6个细胞在1.5ml管中，离心分离细胞，在300×g离心10分钟，弃去上清液并冷冻干燥的颗粒4小时。保持在-80℃直到使用。 </li><li>进行验证试验，重悬细胞完全RPMI（数量取决于进一步的分析是选择），并继续进行步骤4。 </li></ol> 2，双向电泳（2-DE）（图2） 2.1）等电聚焦电泳（IEF） <ol><li>溶解CLL细胞颗粒用380微升2-DE缓冲液（344微升的RBthio解决方案的最终体积（9M尿素，10毫米的Tris，4％CHAPS），30微升65毫米的DTT，6微升2％IPG缓冲两性电解质pH为4-7）。 </li><li>适用于蛋白质样品（1毫克），以18 IPG胶条（pH为4-7），并进行IEF以下标准协议如由帝等人 10 </li><li>补充有新鲜的DTT 2％，在RT15分钟：（50含有6M尿素，30％甘油，2％SDS毫pH为8.8的Tris-HCl缓冲液EB）在2毫升平衡缓冲液中平衡的条。放弃EB + DTT和加入2ml的EB辅以2.5％碘乙酰胺。孵育在室温下15分钟的摇动平台上。 </li></ol> 2.2），SDS-PAGE <ol><li>干在纸条而不触及凝胶去除多余的EB。加载每个条上的9-16％梯度SDS-PAGE凝胶的顶部。加入小体积的SDS-电泳缓冲液（Tris 25mM的，甘氨酸192毫米，SDS 0.1％重量/体积）上的凝胶的顶部，以促进负载的钢带的。 </li><li>加入〜加入5ml琼脂溶液（0.8％），以防止浮带的密封胶，然后组装的电泳单元和填充用SDS-电泳缓冲液中。在4℃下进行运行在60毫安/凝胶4小时。 </li><li>从电泳装置中取出凝胶，并将其放置在适当的染色中。 </li></ol> 2.3）银染色制备凝胶<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td>定影液</td><td> 50％甲醇12％乙酸 0,05％的福尔马林</td><td> 2小时（或过夜）* </td></tr><tr><td>洗涤缓冲液</td><td> 35％Ehanol </td><td> 20分钟（重复步骤三次） </td></tr><tr><td>增敏</td><td> 0,02％硫代硫酸钠</td><td> 2分钟</td></tr><tr><td>洗</td><td> H 2 O的</td><td> 5分钟（重复步骤三次） </td></tr><tr><td>硝酸银</td><td> 0,2％硝酸银0076％福尔马林</td><td> 20分钟</td></tr><tr><td>洗</td><td> H 2 O的</td><td> 1分钟（重复此步骤二次） </td></tr><tr><td>开发者</td><td> 6％的碳酸钠0.0004％硫代硫酸钠 0,05％的福尔马林</td><td>直到染色充足</td></tr><tr><td>停止</td><td> 50％甲醇</td><td> 30分钟</td></tr><tr><td></td><td> *需要2小时的最小时间为蛋白质固定</td><td></td></tr></tbody></table> 表1。 注：蛋白质斑点可以通过凝胶染色与MS兼容的银染11可视化。所有的解决方案所需为银染色列于表1。 <ol><li>制备约200毫升每一溶液/凝胶中，并按照表1中所示，小心地添加各溶液到含有凝胶的染色盒。完成摇摆的平台上所有的孵化。 </li><li>染色后，取出一站式解决方案，并使凝胶1％醋酸溶液，直到采集。 </li></ol> 2.4）凝胶采集与分析<ol><li>使用内染色3天密度仪获得高分辨率的图像。 </li><li>通过使用软件进行2-DE图谱分析2-D蛋白质模式。 注：该软件允许快速和可靠的图像比较。 <ol><li>通过选择“创建新项目”从上下文菜单中创建一个项目。创建一个文件夹并选择“导入凝胶图像”与.TIF，巴纽导入​​凝胶。或.gel扩展。 </li><li>一旦凝胶导入并添加到worksp王牌，通过在菜单中选择凝胶进行当场检测，并选择“编辑&gt;现场&gt;检测”执行自动当场检测。 </li><li>其次，从菜单中选择“减法背景”选择执行背景减除。注意：在此之后就可以比较多个凝胶并检测通过定量和/或定性分析的差异或相似性。 </li></ol></li></ol> 3，蛋白质鉴定通过MALDI-TOF MS分析（图3） 3.1），蛋白质的消化<ol><li>无论是手动（用干净的手术刀切割）或自动切除术用清水冲洗和消费点兴趣凝胶的凝胶。 </li><li>与水（100-150微升）洗凝胶粒子，自旋向下（30秒400 XG）除去液体。 </li><li>加入的CH 3 CN等体积（根据凝胶体积）的凝胶片，并等待10-15分钟，直至凝胶碎片收缩。干凝胶颗粒í呐真空离心机。 </li><li>添加75-100微升10毫米二硫赤藓醇稀释在50毫米的NH 4 HCO 3孵育30分钟，56℃（还原步骤）。再次收缩的凝胶片，用CH 3 CN，如步骤3.1.3中描述。 </li><li>通过加入溶液55 mM的碘乙酰胺稀释于50mM NH 4 HCO 3，孵育20分钟，在室温下在黑暗中的75-100微升进行烷基化。 </li><li>使用足够量如步骤3.1.3中所述，以覆盖凝胶块，用CH 3 CN收缩一次的凝胶片。干式真空离心机。 </li><li>在含25mM NH 4 HCO 3，5mM的氯化钙 ， 和12.5毫微克胰蛋白酶/微升，在4℃下进行20分钟的缓冲液再水合粒子。使用足够量的缓冲来覆盖凝胶块。从凝胶块中取出的未吸收的上清液，加入25 mM的NH 4 HCO 3，5毫米氯化钙2没有胰蛋白酶COV尔的凝胶。 </li><li>离开样品在37℃下至少3小时（所需的肽消化的最短时间）至过夜。 </li></ol> 3.2）质谱分析（干滴法） <ol><li>每个样品到不锈钢MALDI板上的斑点1微升等份和混合用1μl的α-氰基-4 -羟基肉桂酸作为基质，在50％CH 3 CN，0.1％TFA的制备。 </li><li>得到的质谱上的MALDI-TOF质谱仪。累积每点约200光谱，调节激光强度，以防止信号饱和或增加信号。通过数据浏览器软件过程的光谱如下所述： <ol><li>导入dat档，并通过选择“程序&gt;基线校正”进行基线校正。通过选择“程序&gt;质量校正&gt;手动校准校准用胰蛋白酶自溶的产品和基质峰群众;选择峰值接近M / Z 855.1和2163.1 bŸ左拖动并选择相应的参考群众，然后单击“绘图”，并在手动校准窗口“应用校准”。 </li><li>调整阈值的峰值检测（“峰&gt;峰值检测”），重复的活动轨迹（“显示器&gt;复制活跃的踪迹”），选择新的跟踪和执行deisotoping（“峰&gt; deisotoping”）。使用宏“getpeaklist”，产生群众用于识别列表。如果需要，手动删除采摘的高峰清洗名单，并获得更好的识别噪声信号。 </li></ol></li><li>通过搜索用深刻而吉祥物的搜索引擎12进行全面的非冗余蛋白质数据库鉴定蛋白质。 <ol><li>使用以下参数在所有的搜索：一遗漏每肽裂解，氧化蛋氨酸作为修饰的变量，半胱氨酸carbamidomethylation固定的​​修改和initi50ppm的人的质量偏差。 </li></ol></li></ol> 4，细胞骨架的活性测定（图4） 注：将细胞再悬浮于步骤1中的完全RPMI（10 6个细胞/ 200微升）进行纯化。 4.1）迁移实验。该测试可分析的细胞（淋巴细胞）的迁移能力的定量。使用6.5mm的直径和5.0微米的孔径的transwell小室并以一式三份进行测定。在不同类型的细胞的情况下优化孔径和迁移时间（步骤4.1.3）。 <ol><li>通过加入600微升的完全RPMI中具有或不具有特异性刺激（ 即 ，SDF-1）分别测量诱导的自发迁移准备下部腔室。 </li><li>将上部腔室就可以了，并让系统平衡30分钟，在37℃培养箱中。 </li><li>种子10 6细胞/200μl的在上部腔室（总细胞数），并培育在孵化器中的板4小时，在37℃。 </li><li>除去上部腔室，收集在下部腔室中的介质，并用1ml的PBS洗涤下部腔室一次。收集的PBS中的管。 </li><li>离心5分钟300 XG，丢弃在500μl的PBS上清，重悬。 </li><li>通过流式细胞术，计数采集的1分钟后的迁移细胞的数目。在壳体的分离的细胞群，没有必要添加任何表面抗体。查中的双维散点图考虑到细胞的侧散射和可视化在1分钟内，这是用于计算的数目的事件数的细胞数。 </li><li>计算迁移指数（MI）为： <img alt="式（1）" fo:content-width="3.5in" src="/files/ftp_upload/51942/51942eq1.jpg" /></li></ol> 4.2）附着力试验。此测定法允许测量所述细胞的粘附能力。一式三份进行检测。 <ol><li>预涂96 WELLS板（平底）用50μl/孔的任2％BSA-PBS（作为背景）或1微克ICAM-1的稀释于PBS中，过夜，在4℃。 </li><li>用PBS洗涤平板两次，并通过添加2％BSA-PBS（100μl/孔）1小时，在37℃下阻断非特异性位点。 </li><li>在完全RPMI同时悬浮细胞以5×10 6 / ml时，标记他们用1毫米CMFDA绿色电池跟踪器和孵化30分钟，在37℃。 </li><li>加入1×10 6个细胞/孔的预包被的孔中弃去封闭液（步骤4.2.2）后，孵育1小时，在37℃下。 </li><li>用酶标仪定量附着力（激发滤光片：485 nm，发射滤波器：535牛米）。在总输入（INPUT）进行第一次测量。 </li><li>用2％BSA-PBS轻轻洗涤该板（200μl/孔），4倍（倍）。每个清洗步骤（粘附倍）后进行板的读数。 </li><li>经过背景扣除每次测量，计算具体adhes离子（附着力）： <img alt="式（1）" fo:content-width="2in" src="/files/ftp_upload/51942/51942eq2.jpg" /></li></ol> 4.3）聚合试验。这是一个比色测定，量化的F-肌动蛋白聚合。一式三份进行检测。 <ol><li>重悬1×10 ^ 6个细胞于100μl预热的完全RPMI中，并添加特定的刺激（ 即 α-IgM的20微克/毫升）10分钟。 </li><li>停止用400μl的4％多聚甲醛的反应和固定的细胞在室温下10分钟。 </li><li>用PBS（离心机在300×g离心5分钟，4℃）洗涤3次。 </li><li>弃去上清液并通透细胞，在冰上的PBS-甙0.2％（200微升），持续2分钟。 </li><li>洗3次，用PBS-甙0.1％（离心机在100×g离心5分钟，4℃），弃于100μl的PBS-甙0.1％的上清，重悬。 </li><li>为30分钟，在室温吨染色用鬼笔环肽-Alexafluor 488（3微克/毫升）emperature在黑暗中。 </li><li>用PBS-0.1％皂苷洗3次。丢弃在300μL的PBS上清，重悬。 </li><li>量化的F-肌动蛋白的流动量术，计算所述鬼笔环肽的平均荧光的强度（MFI）。为了产生基于荧光强度的直方图中的单个维度的情节所产生的数据进行分析时，MFI值显示在图形报告。 </li><li>测量的F-肌动蛋白聚合（ΔF-肌动蛋白）为： <img alt="式（1）" fo:content-width="2in" src="/files/ftp_upload/51942/51942eq3.jpg" /></li></ol>

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	<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. 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=Monoclonal+B-cell+lymphocytosis+and+chronic+lymphocytic+leukemia.">Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia.</a> N Engl J Med. 359, 575-583 (2008).</li><li>Dagklis, A., Fazi, C., Scarfo, L., Apollonio, B., 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=Monoclonal+B+lymphocytosis+in+the+general+population.">Monoclonal B lymphocytosis in the general population.</a> Leuk Lymphoma. 50, 490-492 (2009).</li><li>Fazi, 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=General+population+low-count+CLL-like+MBL+persists+over+time+without+clinical+progression,+although+carrying+the+same+cytogenetic+abnormalities+of+CLL.">General population low-count CLL-like MBL persists over time without clinical progression, although carrying the same cytogenetic abnormalities of CLL.</a> Blood. 118, 6618-6625 (2011).</li><li>Caligaris-Cappio, F., Bertilaccio, M. 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. S., Thomas, M. E., Mulligan, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocytes,+B+lymphocytes,+and+clonal+CLL+cells:+observations+on+the+impact+of+the+new+diagnostic+criteria+in+the+2008+Guidelines+for+Chronic+Lymphocytic+Leukemia+(CLL).">Lymphocytes, B lymphocytes, and clonal CLL cells: observations on the impact of the new diagnostic criteria in the 2008 Guidelines for Chronic Lymphocytic Leukemia (CLL).</a> Blood. 113, 6496-6497 (2009).</li><li>Conti, 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=Proteome+study+of+human+cerebrospinal+fluid+following+traumatic+brain+injury+indicates+fibrin(ogen)+degradation+products+as+trauma-associated+markers.">Proteome study of human cerebrospinal fluid following traumatic brain injury indicates fibrin(ogen) degradation products as trauma-associated markers.</a> J Neurotrauma. 21, 854-863 (2004).</li><li>Mortz, E., Krogh, T. N., Vorum, H., Gorg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+silver+staining+protocols+for+high+sensitivity+protein+identification+using+matrix-assisted+laser+desorption/ionization-time+of+flight+analysis.">Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis.</a> Proteomics. 1, 1359-1363 (2001).</li><li>Zhang, W., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProFound:+an+expert+system+for+protein+identification+using+mass+spectrometric+peptide+mapping+information.">ProFound: an expert system for protein identification using mass spectrometric peptide mapping information.</a> Anal Chem. 72, 2482-2489 (2000).</li><li>Scielzo, 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=HS1+protein+is+differentially+expressed+in+chronic+lymphocytic+leukemia+patient+subsets+with+good+or+poor+prognoses.">HS1 protein is differentially expressed in chronic lymphocytic leukemia patient subsets with good or poor prognoses.</a> J Clin Invest. 115, 1644-1650 (2005).</li><li>Muzio, 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=HS1+complexes+with+cytoskeleton+adapters+in+normal+and+malignant+chronic+lymphocytic+leukemia+B+cells.">HS1 complexes with cytoskeleton adapters in normal and malignant chronic lymphocytic leukemia B cells.</a> Leukemia. 21 (9), 2067-2070 (2007).</li><li>Scielzo, 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=HS1+has+a+central+role+in+the+trafficking+and+homing+of+leukemic+B+cells.">HS1 has a central role in the trafficking and homing of leukemic B cells.</a> Blood. 116, 3537-3546 (2010).</li><li>Kashuba, E., 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=Proteomic+analysis+of+B-cell+receptor+signaling+in+chronic+lymphocytic+leukaemia+reveals+a+possible+role+for+kininogen.">Proteomic analysis of B-cell receptor signaling in chronic lymphocytic leukaemia reveals a possible role for kininogen.</a> J Proteomics. 91, 478-485 (2013).</li><li>Dhamoon, A. S., Kohn, E. C., Azad, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ongoing+evolution+of+proteomics+in+malignancy.">The ongoing evolution of proteomics in malignancy.</a> Drug Discov Today. 12, 700-708 (2007).</li><li>Ummanni, 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=Identification+of+clinically+relevant+protein+targets+in+prostate+cancer+with+2D-DIGE+coupled+mass+spectrometry+and+systems+biology+network+platform.">Identification of clinically relevant protein targets in prostate cancer with 2D-DIGE coupled mass spectrometry and systems biology network platform.</a> PLoS One. 6, (2011).</li></ol>

The authors have nothing to disclose.

<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">
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		<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>
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		<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?...

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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 ...

from-2de-gel-spot-to-protein-function-lesson-learned-from-hs1-chronic

Research

JoVE Journal

Medicine

13.6K 次观看.  IRCCS, San Raffaele Scientific Institute. 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。 

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

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

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 processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on 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

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this experiment, we demonstrate the administration of low-frequency repetitive TMS (rTMS) to an optimal stimulation site in the right hemisphere in patients with chronic non-fluent aphasia. A battery of standardized language measures is administered in order to assess baseline performance. Patients are subsequently randomized to either receive real rTMS or initial sham stimulation. Patients in the real stimulation undergo a site-finding phase, comprised of a series of six rTMS sessions administered over five days; stimulation is delivered to a different site in the right frontal lobe during each of these sessions. Each site-finding session consists of 600 pulses of 1 Hz rTMS, preceded and followed by a picture-naming task. By comparing the degree of transient change in naming ability elicited by stimulation of candidate sites, we are able to locate the area of optimal response for each individual patient. We then administer rTMS to this site during the treatment phase. During treatment, patients undergo a total of ten days of stimulation over the span of two weeks; each session is comprised of 20 min of 1 Hz rTMS delivered at 90% resting motor threshold. Stimulation is paired with an fMRI-naming task on the first and last days of treatment. After the treatment phase is complete, the language battery obtained at baseline is repeated two and six months following stimulation in order to identify rTMS-induced changes in performance. The fMRI-naming task is also repeated two and six months following treatment. Patients who are randomized to the sham arm of the study undergo sham site-finding, sham treatment, fMRI-naming studies, and repeat language testing two months after completing sham treatment. Sham patients then cross over into the real stimulation arm, completing real site-finding, real treatment, fMRI, and two- and six-month post-stimulation language testing.

We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.

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

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

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

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

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

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

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

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

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

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

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

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

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

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

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

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical activity and longer life expectancy. Despite its increasing prevalence, the underlying pathogenesis still remains unclear, and treatment is generally symptomatic. Recently, numerous therapeutic options, including growth factors, stem cells, and gene therapy, were investigated in hopes of enhancing the healing potency of the degenerative tendon. However, the majority of these research studies were conducted only on animal models or healthy human tenocytes. Despite some studies using pathological tenocytes, to the best of our knowledge there is currently no protocol describing how to obtain human degenerative tenocytes. The aim of this study is to describe a standard protocol for acquiring human degenerative tenocytes. Initially, the tendon tissue was harvested from a patient with lateral epicondylitis during surgery. Then biopsy samples were taken from the extensor carpi radialis brevis tendon corresponding to structural changes observed at the time of surgery. All of the harvested tendons appeared to be dull, gray, friable, and edematous, which made them visually distinct from the healthy ones. Tenocytes were cultured and used for experiments. Meanwhile, half of the harvested tissues were analyzed histologically, and it was shown that they shared the same key features of tendinopathy (angiofibroblastic dysplasia or hyperplasia). A secondary analysis by immunocytochemistry confirmed that the cultured cells were tenocytes with the majority of the cells having positive stains for mohawk and tenomodulin proteins. The qualities of the degenerative nature of tenocytes were then determined by comparing the cells with the healthy control using a proliferation assay or qRT-PCR. The degenerative tenocyte displayed a higher proliferation rate and similar gene expression patterns of tendinopathy that matched previous reports. Overall, this new protocol might provide a useful tool for future studies of tendinopathy.

In vitro use of degenerative tenocytes is essential when investigating the efficacy of novel treatment on tendinopathy. However, most research studies use only the animal model or a healthy tenocyte. We propose the following protocol to isolate human degenerative tenocytes during surgery.

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 ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

一种生成周期负荷诱导的大鼠膝关节内软骨病变模型的非侵入性方法

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...