这项工作得到了补助金从La伊夫基金会Cotrel的DE L&#39;法兰西学院，巴黎，法国（以莫罗博士）的支持，健康研究加拿大学院（授予PP2-99466到莫罗博士），并从第四维脊椎有限责任公司，New York，美国（研究经费莫罗博士）。

整个过程进行无菌生物引擎盖下，所有的解决方案和设备的进入与细胞的接触必须是无菌的。 1。基本溶液的制备<ol><li>根据表1制备溶液。 </li><li>保持平衡盐溶液（BSS）在室温和所有其他的解决方案，在4℃直至使用时。 </li><li>保暖防寒媒体37℃的水浴使用前几分钟。 </li></ol> 2。外周血单个核细胞的制备和储存<ol><li>收集10毫升全血EDTA处理的收集管，用5毫升每一等分准备的外周血单个核细胞两等份。 </li><li>传递从EDTA处理收集管到50ml管5毫升全血。 </li><li>添加BSS等体积和轻柔吹打混匀样品上下。 </li><li>将3毫升聚蔗糖的两个15毫升猎鹰管。 </li><li>仔细层4.5毫升稀释的血液混合物在菲可在每个管中。 </li><li>让管休息长达5分钟有利于血液和聚蔗糖的明确分工。 </li><li>离心管中，在400×g离心30分钟，在室温下用不加制动。 </li><li>小心地从离心分离机取出管子，以便不打扰分层。的是外周血单个核细胞在BSS /聚蔗糖界面可见。 </li><li>收获PBMC中的混浊层在两个管与吸管的接口和转移到新的50ml管中。 </li><li>加20 ml完全介质。 </li><li>离心管中于288×g下在室温下7分钟。 </li><li>吸去上清。 </li><li>重悬细胞沉淀在500微升补充媒体。 </li><li>加冷冻培养基等体积的。 </li><li>细胞悬液转移至离心管。 </li><li>将离心管与异丙醇的cryofreezing容器。 </li><li>储存在冷冻容器中，在-80℃过夜。 </li><li>风帆ansfer冷冻的PBMC分装到液氮中用于长期贮存。 </li></ol> 3。功能分析 1。第1天<ol><li>放置等分试样从液氮在水浴中于37℃一分钟，或直至解冻。 </li><li>细胞悬液转移至50ml管中，用无菌移液管。 </li><li>加15 ml完全介质和自旋细​​胞下降，在200×g离心5分钟，在室温下。 </li><li>吸去上清。 </li><li>轻轻悬浮细胞沉淀1毫升PHA媒体。 </li><li>完成体积至20ml用相同的介质。 </li><li>盖上管松散，以允许空气进入。 </li><li>离开管过夜，在37℃的CO 2培养箱，让静态的淋巴细胞转化为快速增殖淋巴母细胞。 </li></ol> 2。 2天<ol><li>采取管从孵化器，完全拧紧瓶盖和自旋向下的细胞在200×g离心5分钟在室温下进行。 </li><li>吸去上清。 </li><li>轻轻悬浮细胞沉淀1毫升完整的媒体。 </li><li>完成体积至20ml用相同的介质。 </li><li>盖上管松散，以允许空气进入。 </li><li>离开该管过夜，于37℃在CO 2培养箱中扩大细胞数量。 </li></ol> 3。 3天<ol><li>得到的管出来的培养箱，完全拧帽并旋转向下细胞在200×g离心5分钟，在室温下进行。 </li><li>吸去上清。 </li><li>两次用10ml的RPMI-1640通过离心，在200×g离心5分钟，在室温下洗涤​​细胞。 </li><li>轻轻重悬细胞沉淀在600μLRPMI-1640。 </li><li>测定细胞浓度和活力，用自动细胞计数器和活力分析仪。 </li><li>加的RPMI-1640的适当体积，并调整到1.5×10 5 cells/20微升的细胞浓度。 </l我&gt; <li>在1.5ml Eppendorf管中的细胞治疗用重组OPN（rOPN）或载体（PBS）。 <ol><li>转移100微升细胞悬液的两个无菌1.5ml离心管中。 </li><li>在一试管中加入rOPN至0.5微克/毫升的最终浓度。 </li><li>添加PBS，在第二管的等体积。 </li><li>轻轻地上下吹打使用无菌移液管套在100μl的两次混合各条件。 </li></ol></li><li>制备小样品的96孔微量培养板。 <ol><li>添加5微升的RPMI-1640的每个孔中。 </li><li>离心板在200×g离心3分钟以除去任何气泡。 </li></ol></li><li>种子未经处理的细胞，以及与rOPN或PBS处理的细胞。 <ol><li>在从管细胞转移到微孔板，轻轻吹吸一次，以确保细胞均匀悬浮液。 </li><li>加入40微升每孔细胞悬液一式四份对于未处理的细胞，一式两份的rOPN或PBS处理的细胞。回复FER 图1的设计。这样的设计使12名患者被在相同的微板进行测试。 </li><li>离开细胞板的无菌罩下5分钟，以让细胞放置在培养箱中之前休息和定居均匀到井的底部。 </li></ol></li><li>孵育板18小时，于37℃在CO 2培养箱中以优化的OPN的效果。 </li></ol> 4。 4天<ol><li>与化合物运行的板块。 <ol><li>就拿盘开出的孵化器，让它在室温下约30分钟。 </li><li>通过在990微升的RPMI-1640中加入10μl储备溶液（10毫摩尔）的制备将1ml100μM的促生长素抑制素和异丙基肾上腺素在RPMI-1640中。 </li><li>通过分配20μl，以适当的孔中， 如图2所示填充复合板。 </li><li>用预切刺穿密封件覆盖的复合板，以避免改变化合物的浓度，由于蒸发前或孵化在CDS为基础的系统中。 </li><li>负载单元板，移液器吸头，复合板进入CDS为基础的系统。 </li><li>在CDS为基础的工具软件名称的板块。 </li><li>选择适当的协议。编辑与外周血单个核细胞分类的协议被称为“受体激动剂非贴壁细胞小试板RT 15分钟”。转到协议框中，在协议列表中选择此协议</li><li>单击“开始”启动协议。 </li><li>集成的流体系统通过注射，每孔5μl的实现中的50微升的总体积为10μM的终浓度同时增加了所述化合物的所有的孔中。 </li><li>在CDS为基础的系统自动收集的数据为化合物加入后15分钟。 </li></ol></li><li>数据分析<ol><li>选择的频率的低和高范围的计算对于非提取的值时使用贴壁细胞。 </li><li>选择漂移校正，可校正随着时间的推移在基线阻抗测量的线性变化。 </li><li>选择数据过滤，以减少由于电子噪声和复合另外变化的动力学响应测量。 </li><li>选择最大 - 最小方法为全面分析时间。 </li><li>数据导出到Excel中下盘的格式选项。 </li><li>通过从响应幅度GS刺激（RMGS）使用下列公式计算的平均值中减去响应幅值的平均值在GI刺激（RMGI）计算增量G（ΔG）： ΔG= RMGI - RMGS </li><li>计算的OPN对由响应幅值的平均值除以在GI刺激OPN（RmGiOPN）与响应幅值的平均值存在于胃肠道的刺激在PBS中的存在游戏王介导的应答的折叠效果（Fe）的比例（ RmGiPBS）使用下列公式计算： FE = 100×（RmGiOPN / RmGiPBS） </li><li>指T2能够进行分类的患者。 </li></ol></li></ol>

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这项工作导致多项专利由圣 - 和Justine大学医院以及许多其他持有正等待在几个国家。第四维脊柱有限责任公司是圣 - 和Justine大学医院的独家许可。

我们已经描述了基于细胞的预后测试为特发性脊柱侧凸了适用于外周血单核细胞（PBMC）新鲜分离的或保守的急冻长达一年在液氮中的详细过程。由于测试大量的个人在使用新鲜分离的外周血单个核细胞是繁琐，程序提交了冷冻外周血单个核细胞，提供在临床上较为实际的选择。然而，存在的问题与解冻后细胞聚集时，冷冻的PBMC与最初使用时，有时会导致测定间变异遇到。为了最大限度地提高测定的再现性，建议避免冻融循环和仅使用一次的冷冻样品。该过程是很简单的，允许精确检测在短时间内有缺陷的Gi蛋白的功能。使用此程序，无症状和侧凸儿童可以很容易地划分，以更好地预测其临床结果不为他们的健康有任何危险。何WEVER，根据在GI刺激相对于健康对照组12最大响应程度进行分类时，应满足的对照组几个要求。事实上，为了有一个适当的比较队列，控制对象必须是年龄和性别匹配的，不会对任何种类的药物，并提供私人信息，如过去的个人/家族病史。这些要求可能构成对控制对象的招聘的一个重要障碍。因此，通过检查不平衡响应于Gi和Gs蛋白刺激在同一个体之间的相似度进行分类是理想的，以消除使用对照受试者的必要性。 使用CDS为基础的系统来执行此测试的预后是在同时提供游戏王和GS-介导的细胞应答在相同的测定条件显著。尽管许多患者可分为无时biguity使用动态范围定为这个实验中，有少数病人会在范围的边缘表现出值，就说明了本报告中的结果。歧视这些人，我们通过证明OPN诱导的三官能团之间的游戏王介导的细胞反应差效应引入的OPN效果的评估上反应在GI刺激。事实上，我们发现，在骨桥蛋白，响应于游戏王刺激增加FG1，的存在，而它在FG2和FG3减小，在FG2更高的程度。尽管OPN成本高，使用这种趋化因子是十分必要的区分不明确的情况下，因此提高我们的分类分析的准确性。然而，这种试剂盒中，最低每孔1.5×10 5个细胞的需要我们的实验条件下观察到的细胞反应和CDS为基础的系统是不利的。某些患者样品不会有足够数量的细胞进行测试。在这种情况下，有必要回顾一下病人额外的血液采集，它可以是令人担忧的家庭和令人沮丧的医疗和实验室工作人员。前瞻性研究计划在我们的实验室来解决这个问题。 然而，目前的协议依赖于简单而行之有效的方法来制备细胞，而测试是使用经过验证的无标记系统13，14来监测细胞的反应自动化。用时可用于其他疾病的预后遗传平台相比，测试平台是相对便宜的。此外，所有的一次性材料，包括血液收集管，技巧，特殊电极微孔板，和圆锥形和Eppendorf管的成本，是不是很昂贵，估计少于3,000元，完成约千术测试。虽然这个估计不包括样品制备人工成本并进行测试，这个测试仍然大大高于新一代测序平台的更多实惠。值得注意的是，不仅是成本比较遗传平台上，但更具体地涉及到AIS的领域中，是关联到辐射成像的费用。今天，在美国超过一百万的儿童和在加拿大大约10万名儿童被诊断为特发性脊柱侧凸，以及诊断和监测脊柱侧凸儿童用X射线照射的总成本每年超过250十亿美元在北美。因此，我们的细胞为基础的检测程序，预计将适用于常规筛查和监测儿童特发性脊柱侧凸没有健康风险，并以更低的成本。

特发性脊柱侧弯是一种原因不明的脊柱畸形一般定义为侧向弯曲大于10度伴有椎体旋转1。这种状况影响了儿科人口的4％，是9至13岁2,3,4岁之间最常见的诊断。诊断主要是一种排斥，只有在排除了脊柱畸形的其他原因，如椎畸形，神经肌肉综合征或疾病作出。传统上，trunkal不对称性是由亚当斯向前弯曲试验发现，并在体检5与测量计测量。诊断然后可通过放射线照相观测的曲线，并使用科布方法6中的角度测量来确认。 一旦确诊，医生在管理侧凸儿童主要关注的是曲线是否会取得进展。事实上，侧凸进展往往是不可预测的，比7男生更频繁地在女孩中观察到。如果不经治疗，曲线可以显着进步，创造显著身体畸形，甚至心肺的问题。这些表现成为当曲线超过70°8,9危及生命。目前的治疗方案，以防止或阻止侧凸进展包括支撑和手术治疗。在一般情况下，支撑被推荐用于在25-40°之间的曲线，而手术是保留曲线大于45°或不响应支撑。 约，确诊为特发性脊柱侧弯的孩子10％有曲线进展，需要矫正手术10。目前，还没有有效的方法或测试可用来识别这类病人。因此，所有确诊儿童受到多重X光片在数年内，通常直到他们到达骨骼成熟。据估计，在典型患者的脊柱侧凸将有约22放射学检查在3年的时间11。由于多个影像学检查的潜在风险，替代方法，可以让不暴露孩子于电离辐射进行特发性脊柱侧凸的预后是非常可取的。随着满足这一需求的意向，我们以前开发一个基于细胞的筛选试验作为预后测试越早确定无症状儿童在发展特发性脊柱侧凸的风险。该测试预计在无症状和影响的儿童的临床结果通过检查Gi蛋白的功能状态，并根据对Gi蛋白刺激12最大响应程度由CDS衡量孩子分成三官能团（FG1，FG2和FG3）为基础的系统。这个系统主要是用来通过G蛋白在各种细胞类型13,14,15,16，以评估信号转导。它产生有关的信息细胞通过以下细胞表面受体的活化测量阻抗的变化总集成响应于外部刺激。细胞被接种到微孔板包含电极的孔的底部，并且系统施加一个小电压诱发的细胞外和跨细胞电流。下列化合物注入到井中，细胞表面受体被刺激和信号转导事件的发生，从而导致影响细胞外和跨细胞电流的流动细胞的变化，进而影响测量的幅度。通过这种方法，脊柱侧凸患者和儿童更是发展脊柱侧弯的风险是当与健康对照组相比，较不响应能力Gi蛋白的刺激，而分类是基于减少相对于对照组的程度的百分比。该分类范围是固定的10至40％为FG3，40和FG2 60％，60至90％为FG1 12。 17点的风险。这种分类测试的精度还进一步通过在响应中的官能团游戏王刺激显示出骨桥蛋白（OPN）的差动作用得到改善。 在这里，我们介绍的是目前在我们的实验室进行这种功能测试的实验和分析程序的详细步骤。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Wisent, Inc</td>
			<td>350-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Therno Scientific Hyclone</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Plaque Plus</td>
			<td>GE Healthcare</td>
			<td>17144003</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Invitrogen</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytohemagglutinin</td>
			<td>Invitrogen (Gibco)</td>
			<td>10576-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Osteopontin</td>
			<td>R&#38;D Systems, Inc</td>
			<td>1433-OP/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Somatostatin</td>
			<td>Tocris</td>
			<td>1157</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproterenol</td>
			<td>Tocris</td>
			<td>1743</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Wisent, Inc</td>
			<td>311-010-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipette tips</td>
			<td>Axygen Scientific</td>
			<td>301-06-451</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Eppendorf tubes</td>
			<td>Ultident</td>
			<td>24-MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>VWR International</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellkey small sample 96W microplate</td>
			<td>Molecular Devices</td>
			<td>1026496</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellkey tips</td>
			<td>Cybio</td>
			<td>OL3800-25-559N</td>
			<td></td>
		</tr>
		<tr>
			<td>Precut pierceable seals</td>
			<td>Excel Scientific, Inc</td>
			<td>XP-100</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Vicell XR</td>
			<td>Beckman Coulter</td>
			<td>731050</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>Cell culture hood</td>
			<td>Forma Scientific</td>
			<td>1284</td>
			<td>Class II</td>
		</tr>
		<tr>
			<td>Liquid nitrogen storage</td>
			<td>Thermo Scientific</td>
			<td>CY5093570</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VWR International</td>
			<td>89032-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard light microscope</td>
			<td>Leica Microsystems</td>
			<td>DMIL LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Thermo Scientific</td>
			<td>51019557</td>
			<td>5% CO2 at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Low speed centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004364</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellkey system</td>
			<td>Molecular Devices</td>
			<td>1019185</td>
			<td>CDS-based instrument</td>
		</tr>
	</tbody>
</table>

cell-based assay protocol for the prognostic prediction of idiopathic scoliosis using cellular dielectric spectroscopy

这个协议的细节的实验和分析方法在我们实验室开发的作为功能性测试来预测无症状和受影响儿童特发性脊柱侧凸的预后基于细胞的测定法。该法由Gi和GS通过细胞介电谱（CDS）的蛋白质在外周血单个核细胞（PBMC）的功能状态的评价，使用自动化的CDS为基础的仪器，和儿童的分类成三个官能团（FG1 ，FG2，FG3）相对于不平衡响应于Gi和GS蛋白刺激的程度之间的轮廓。该分类是由骨桥蛋白（OPN）对反应组和疾病的严重恶化被引用FG2游戏王之间的刺激效果差进一步证实。大约为10毫升的血液的体积，需要通过Ficoll梯度和细胞中提取的PBMC，然后在液氮中保存。 P的足够数量的骨髓细胞进行检测后，被细胞培养两天获得的。本质上，将细胞先孵育phytohemmaglutinin（PHA）。经过24小时培养后，培养基被替换为一个的PHA的无培养基进行24小时前，细胞接种和OPN治疗。细胞，然后光谱筛选他们的反应，生长抑素和异丙肾上腺素，通过其同源受体分别激活Gi和GS蛋白。既生长抑素及异丙肾上腺素同时注入带有集成流体系统和细胞的响应被监测15分钟。该法可以用新鲜或冷冻的外周血单个核细胞进行，并且该过程在4天内完成。

细胞活力是可比较在所有的86和96％的范围内一致的样本值中。与此相反，高的变化，指出在细胞数的样品（ 表3）中。所用的32个样本中，2例细胞的数量不足和没有被分类。功能分类的依据Gi和GS信令之间的不平衡的程度的结果的一个例子显示于图3。该图中的垂直轴被分为三段划分功能组具有动态范围确定为&gt; 10为FG3，10和-10之间为FG2，最后&lt;-10为FG1。其中30例患者在这里进行测试，14，6，5例患者进行了明确划分为FG3，FG2，和FG1，分别，而五名患者，尤其是345，353，370，371，和382，均在范围边界。对反应在GI刺激OPN效果评价时曾透露，OPN增加响应的PA患者353和371。与此相反，响应减少了患者345和382的50％以上，并通过在患者370以下rOPN治疗小于50％。所以，根据我们的分类标准（ 表2），我们能够进行分类的患者353与371中FG1，病人345和FG2 382，并在FG3病人370。并联，所有患者进行了筛选，其响应于Gi蛋白刺激和对照受试者相比。正如预期的那样，所有患者均低于对照组不太敏感，并通过我们的新程序划分在同一功能组的患者表现出的最大响应（ 图5）相似的水平。此外，每个功能组的患者之间的差距是与我们传统的范围分类12一致，验证了我们新的程序。脊柱侧凸患者定期随访中我们在圣 - 和Justine医院特诊的大型队列的分类显示，三官能团被类似地分布在适中的情况下，而FG2为优势之间严重的情况下（ 图6），确定患者分为该官能团为更危险的疾病的严重恶化，并表明此分类测试可以在有用预后特发性脊柱侧凸。 <table border="1"><tbody><tr><td rowspan="5">溶液A </td><td>无水D-葡萄糖</td><td> 0.1％ </td></tr><tr><td>氯化钙2·2H 2 O </td><td> 0.05毫米</td></tr><tr><td> 氯化镁 </td><td> 0.98毫米</td></tr><tr><td>氯化钾</td><td> 5.4毫米</td></tr><tr><td>三</td><td> 145毫米</td></tr><tr><td>溶液B </td><td>氯化钠</td><td> 140毫</td></tr><tr><td rowspan="2">平衡盐溶液（BSS） </td><td>溶液A </td><td> 1卷</td></tr><tr><td>溶液B </td><td> 9卷</td></tr><tr><td跨行=“3”&gt;完整的媒体</td><td>的RPMI-1640 </td><td> 500毫升</td></tr><tr><td>抗生素，抗真菌</td><td> 1％ </td></tr><tr><td> FBS </td><td> 10％ </td></tr><tr><td rowspan="3">补充媒体</td><td>的RPMI-1640 </td><td> 50毫升</td></tr><tr><td>抗生素，抗真菌</td><td> 1％ </td></tr><tr><td> FBS </td><td> 40％ </td></tr><tr><td rowspan="4">冻结媒体</td><td>的RPMI-1640 </td><td> 50毫升</td></tr><tr><td>抗生素，抗真菌</td><td> 1％ </td></tr><tr><td> FBS </td><td> 40％ </td></tr><tr><td> DMSO </td><td> 20％ </td></tr><tr><td rowspan="4"> PHA媒体</td><td>的RPMI-1640 </td><td> 500毫升</td></tr><tr><td>抗生素，抗真菌</td><td> 1％ </td></tr><tr><td> FBS </td><td> 10％ </td></tr><tr><td>植物血凝素</td><td> 1％ </td></tr></tbody></table> 表1。基本解决方案。 <table border="1"><tbody><tr><td> 与ΔG动态范围 </td><td> 官能团 </td><td> 与铁动态范围 </td></tr><tr><td> ΔG&lt;-10 </td><td> FG1 </td><td>铁&gt; 100％ </td></tr><tr><td> -10 &lt;ΔG&lt;10 </td><td> FG2 </td><td>铁&lt;50％ </td></tr><tr><td> ΔG&gt; +10 </td><td> FG3 </td><td> 50％&lt;铁&lt;95％ </td></tr></tbody></table>的官能团，根据与ΔG和Fe建立动态范围表2。分类。 <table border="1"><tbody><tr><td> 患者 </td><td> 存活率（％） </td><td> 细胞浓度（×10 6 / ml）的 </td><td> 评论 </td></tr><tr><td> 343 </td><td> 88.7 </td><td> 11.64 </td><td></td></tr><tr><td> 344 </td><td> 90.5</td><td> 13.6 </td><td></td></tr><tr><td> 345 </td><td> 94.4 </td><td> 8.54 </td><td></td></tr><tr><td> 346 </td><td> 94.3 </td><td> 25.79 </td><td></td></tr><tr><td> 347 </td><td> 94.2 </td><td> 27.36 </td><td></td></tr><tr><td> 348 </td><td> 94.6 </td><td> 8.52 </td><td></td></tr><tr><td> 349 </td><td> 91.2 </td><td> 0.82 </td><td>细胞数量不足</td></tr><tr><td> 350 </td><td> 90.3 </td><td> 8.92 </td><td></td></tr><tr><td> 352 </td><td> 92.6 </td><td> 8.28 </td><td></td></tr><tr><td> 353 </td><td> 91.3 </td><td> 12.75 </td><td></td></tr><tr><td> 354 </td><td> 86.9 </td><td> 7.62 </td><td></td></tr><tr><td> 355 </td><td> 91.2 </td><td> 7.51 </td><td></td></tr><tr><td> 356 </td><td> 90.3 </td><td> 9.36 </td><td></td></tr><tr><td> 358 </td><td> 95.1 </td><td> 16.94 </td><td></TD&gt; </tr><tr><td> 359 </td><td> 92.3 </td><td> 13.89 </td><td></td></tr><tr><td> 360 </td><td> 89.4 </td><td> 7.67 </td><td></td></tr><tr><td> 361 </td><td> 93.5 </td><td> 7.84 </td><td></td></tr><tr><td> 365 </td><td> 86.5 </td><td> 2.2 </td><td>细胞数量不足</td></tr><tr><td> 368 </td><td> 92.6 </td><td> 15.69 </td><td></td></tr><tr><td> 369 </td><td> 93.4 </td><td> 10.9 </td><td></td></tr><tr><td> 370 </td><td> 92.5 </td><td> 19.93 </td><td></td></tr><tr><td> 371 </td><td> 88.8 </td><td> 10.68 </td><td></td></tr><tr><td> 374 </td><td> 93.9 </td><td> 16.86 </td><td></td></tr><tr><td> 376 </td><td> 92.9 </td><td> 15.67 </td><td></td></tr><tr><td> 377 </td><td> 93.1 </td><td> 9.99 </td><td></td></tr><tr><td> 378 </td><td> 93.6 </td><td> 13.57 </td><td></td></tr><tr><td> 379 &lt;/ TD&gt; <td> 92.6 </td><td> 19.86 </td><td></td></tr><tr><td> 380 </td><td> 91.1 </td><td> 8.46 </td><td></td></tr><tr><td> 381 </td><td> 93.9 </td><td> 14.82 </td><td></td></tr><tr><td> 382 </td><td> 92.1 </td><td> 23.06 </td><td></td></tr><tr><td> 383 </td><td> 92.9 </td><td> 11.82 </td><td></td></tr><tr><td> 384 </td><td> 89.1 </td><td> 7.73 </td><td></td></tr></tbody></table> 表3。 ％的生存力和细胞浓度为使用自动细胞计数器及活力分析仪确定。 <img alt="图1" src="/files/ftp_upload/50768/50768fig1.jpg" /> 图1。设计用于细胞接种 。 <img alt="图2" src="/files/ftp_upload/50768/50768fig2.jpg" /> 图2。设计用于分配的化合物 。 <img alt="图3" src="/files/ftp_upload/50768/50768fig3.jpg" /> 图3。动态范围使用CDS为基础的系统的功能分类 。图说明反应Gi和患者的特发性脊柱侧凸的外周血单个核细胞获得GS刺激之间的不平衡程度的值。值是由CDS为基础的系统在响应10μM生长抑素及异丙肾上腺素测定。每个点代表在重复的两种反应的ΔG。 <img alt="图4" src="/files/ftp_upload/50768/50768fig4.jpg" /> 图4。rOPN对反应在GI刺激外周血单个核细胞 。在存在或不存在的0.5微克/毫升rOPN细胞进行血清饥饿18小时，然后用10刺激<html> 56，生长抑素M来启动游戏王介导的细胞免疫应答。图中的数据是从最大 - 最小阻抗产生的，并且对应于响应的一式两份的平均值。 <img alt="图5" src="/files/ftp_upload/50768/50768fig5.jpg" /> 图5。Gi蛋白在对照组和scoliostic受试者外周血单个核细胞功能状态 。从对照组和脊柱侧凸患者外周血单个核细胞暴露于增加生长抑素的浓度通过内源性生长抑素受体刺激Gi蛋白。的细胞应答是如在程序段描述的CDS为基础的系统测量。从最大最小阻抗生成曲线。每条曲线代表了非线性回归。数据标准化为在从对照受试者的细胞最大应答和每个点对应于响应的一式两份的平均值。/ 50768/50768fig5large.jpg“目标=”_blank“&gt;点击这里查看大图。 <img alt="图6" src="/files/ftp_upload/50768/50768fig6.jpg" /> 图6。脊柱侧弯的不同阶段之间的分布官能团 。与794中度（10-44°之间的曲率）和162重度（曲率半径大于45°）情况下，定期于圣 - 和Justine医院的大型队列研究脊柱侧凸患者，根据其失衡程度进行分类响应Gi和GS之间刺激。反应是由CDS为基础的系统在响应10μM生长抑素及异丙肾上腺素的测定。

Watch this Scientific Journal Video about Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy at JoVE.com

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

结构化协议提出了一个基于细胞的检测作为一个功能测试使用的细胞介电谱（CDS）来预测特发性脊柱侧凸的预后。该测定法可以与新鲜或冷冻的外周血单核细胞（PBMC）来执行，该过程是在4天内完成。

特发性脊柱侧弯的使用蜂窝介电谱的预后预测细胞为基础的检测协议

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

cell-based-assay-protocol-for-prognostic-prediction-idiopathic

Research

JoVE Journal

Medicine

10.5K 次观看.  Sainte-Justine University Hospital Research Center. 结构化协议提出了一个基于细胞的检测作为一个功能测试使用的细胞介电谱（CDS）来预测特发性脊柱侧凸的预后。该测定法可以与新鲜或冷冻的外周血单核细胞（PBMC）来执行，该过程是在4天内完成。 

视频: Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP)

Many women undergo cesarean delivery without problems, however some experience significant pain after cesarean section. Pain is associated with negative short-term and long-term effects on the mother. Prior to women undergoing surgery, can we predict who is at risk for developing significant postoperative pain and potentially prevent or minimize its negative consequences? These are the fundamental questions that a team from the University of Washington, Stanford University, the Catholic University in Brussels, Belgium, Santa Joana Women's Hospital in S&atilde;o Paulo, Brazil, and Rambam Medical Center in Israel is currently evaluating in an international research collaboration. The ultimate goal of this project is to provide optimal pain relief during and after cesarean section by offering individualized anesthetic care to women who appear to be more 'susceptible' to pain after surgery.
A significant number of women experience moderate or severe acute post-partum pain after vaginal and cesarean deliveries. 1 Furthermore, 10-15% of women suffer chronic persistent pain after cesarean section. 2 With constant increase in cesarean rates in the US 3 and the already high rate in Brazil, this is bound to create a significant public health problem. When questioning women's fears and expectations from cesarean section, pain during and after it is their greatest concern. 4 Individual variability in severity of pain after vaginal or operative delivery is influenced by multiple factors including sensitivity to pain, psychological factors, age, and genetics. The unique birth experience leads to unpredictable requirements for analgesics, from 'none at all' to 'very high' doses of pain medication. Pain after cesarean section is an excellent model to study post-operative pain because it is performed on otherwise young and healthy women. Therefore, it is recommended to attenuate the pain during the acute phase because this may lead to chronic pain disorders. The impact of developing persistent pain is immense, since it may impair not only the ability of women to care for their child in the immediate postpartum period, but also their own well being for a long period of time.
In a series of projects, an international research network is currently investigating the effect of pregnancy on pain modulation and ways to predict who will suffer acute severe pain and potentially chronic pain, by using simple pain tests and questionnaires in combination with genetic analysis. A relatively recent approach to investigate pain modulation is via the psychophysical measure of Diffuse Noxious Inhibitory Control (DNIC). This pain-modulating process is the neurophysiological basis for the well-known phenomenon of 'pain inhibits pain' from remote areas of the body. The DNIC paradigm has evolved recently into a clinical tool and simple test and has been shown to be a predictor of post-operative pain.5 Since pregnancy is associated with decreased pain sensitivity and/or enhanced processes of pain modulation, using tests that investigate pain modulation should provide a better understanding of the pathways involved with pregnancy-induced analgesia and may help predict pain outcomes during labor and delivery. For those women delivering by cesarean section, a DNIC test performed prior to surgery along with psychosocial questionnaires and genetic tests should enable one to identify women prone to suffer severe post-cesarean pain and persistent pain. These clinical tests should allow anesthesiologists to offer not only personalized medicine to women with the promise to improve well-being and satisfaction, but also a reduction in the overall cost of perioperative and long term care due to pain and suffering. On a larger scale, these tests that explore pain modulation may become bedside screening tests to predict the development of pain disorders following surgery.

An Experimental Paradigm for the Prediction of Post-Operative Pain PPOP

Diffuse noxious inhibitory control, temporal summation and wound hyperalgesia testing are demonstrated in the obstetric patient. These tests evaluate inhibitory and excitatory mechanisms of pain processing and are here utilized to evaluate endogenous analgesia at different time-points during pregnancy and the peripartum period to help reveal individual s risk for persistent pain.

用于预测手术后的疼痛的实验范式（PPOP）

Many women undergo cesarean delivery without problems, however some experience significant pain after cesarean section. Pain is associated with negative ...

科研

医学

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

注重形成：一个基于Cell的检测，以确定一个基因的致癌性

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

弥漫性光学光谱治疗急性电离辐射引起的皮肤毒性的定量评价使用小鼠模型

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

使用分体路西酶测定系统识别 HBx-DDB1 相互作用的抑制剂

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently ...

Quantification of Optic Nerve Cross Sectional Area on MRI: A Novel Protocol using Fiji Software

Optic nerve assessment is an important aspect of glaucoma diagnosis and follow-up. This project describes a protocol for a unified methodology of optic nerve cross-sectional assessment and quantification using 3 T MRI for image acquisition and ImageJ's Fiji software for image processing quantification. Image acquisition was performed using 3 T MRI, with proper instructions for the patient to ensure straight fixation during imaging. A T2-weighted fat suppressed sequence was used. A coronal cut taken 3 mm behind the globe and perpendicular to the optic nerve axis should be uploaded to the software. Using the threshold function, the white matter area of the optic nerve is selected and quantified, thus, eliminating inter-individual measurement bias. We also described the normal limits for the optic nerve cross-sectional area according to age, based on previously published literature. We used the described protocol to assess optic nerve of a suspected glaucoma patient. The optic nerve cross-sectional area was found to be within the normal limits, a finding further confirmed via optical coherence tomography of the optic nerve.

We provided a detailed protocol for a standardized method of optic nerve assessment and quantification using MRI, utilizing a widely available imaging sequence, and open access software for image analysis. Following this standardized protocol would provide meaningful data for comparison between different patients and different studies.

MRI 上光学神经横截面区域的量化：使用斐济软件的新协议

Optic nerve assessment is an important aspect of glaucoma diagnosis and follow-up. This project describes a protocol for a unified methodology of optic ...

A Real-World High-Intensity Interval Training Protocol for Cardiorespiratory Fitness Improvement

High-Intensity Interval Training (HIIT) has emerged as an interesting time-efficient approach to increase exercise adherence and improve health. However, few studies have tested the efficiency of HIIT protocols in a &#34;real world&#34; setting, e.g., HIIT protocols designed for outdoor spaces without specialized equipment. This study presents a &#34;real world&#34; training protocol, named &#34;beep training&#34;, and compares the efficiency of a HIIT regiment versus a traditional long-duration Moderate-Intensity Continuous Training (MICT) regiment using this beep training protocol on VO2 max of overweight untrained men. Twenty-two subjects performed outdoor running with MICT (n = 11) or HIIT (n = 11). Cardiorespiratory fitness was assessed before and after training protocols using a metabolic analyzer. Both training protocols were performed 3 days a week for 8 weeks using the Beep Test results. The MICT group performed the exercise program at 60%-75% of the maximum speed of the 20 m shuttle test (Vmax) and with a progression of the distance of 3,500-5,000 m. The HIIT group performed the interval exercise with 7-10 bouts of 200 m at 85%-100% of the maximum speed of the 20 m shuttle test (Vmax), interspersed with 1 min of passive recovery. Although the HIIT group presented a significantly lower training volume than the MICT group (p &#60; 0.05) after 8 weeks of beep training, HIIT was superior to MICT in improving VO2 max (MICT: ~4.1%; HIIT: ~7.3%; p &#60; 0.05). The &#34;real world&#34; HIIT regiment based on beep training protocol is a time-efficient, low-cost, and easy-to-implement protocol for overweight untrained men.

This study presents a low-cost and easy-to-implement "real world" high-intensity interval training (HIIT) protocol for scientific research and discusses its efficiency for cardiorespiratory fitness.

一种用于改善心肺健康的真实世界高强度间歇训练方案

High-Intensity Interval Training (HIIT) has emerged as an interesting time-efficient approach to increase exercise adherence and improve health. However, ...

Network Pharmacology Prediction and Metabolomics Validation of the Mechanism of Fructus Phyllanthi against Hyperlipidemia

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug against hyperlipidemia in Traditional Chinese Medicine (TCM) and Indian Medicine theories, however the potential mechanism requires further exploration. The present research aims to reveal the mechanism of FP against hyperlipidemia based on an integrated strategy combining network pharmacology prediction with metabolomics validation. A high-fat diet (HFD)-induced mice model was established by evaluating the plasma lipid levels, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Network pharmacology was applied to find out the active ingredients of FP and potential targets against hyperlipidemia. Metabolomics of plasma and liver were performed to identify differential metabolites and their corresponding pathways among the normal group, model group, and intervention group. The relationship between network pharmacology and metabolomics was further constructed to obtain a comprehensive view of the process of FP against hyperlipidemia. The obtained key target proteins were verified by molecular docking. These results reflected that FP improved the plasma lipid levels and liver injury of hyperlipidemia induced by a HFD. Gallic acid, quercetin, and beta-sitosterol in FP were demonstrated as the key active compounds. A total of 16 and six potential differential metabolites in plasma and liver, respectively, were found to be involved in the therapeutic effects of FP against hyperlipidemia by metabolomics. Further, integration analysis indicated that the intervention effects were associated with CYP1A1, AChE, and MGAM, as well as the adjustment of L-kynurenine, corticosterone, acetylcholine, and raffinose, mainly involving tryptophan metabolism pathway. Molecular docking ensured that the above ingredients acting on hyperlipidemia-related protein targets played a key role in lowering lipids. In summary, this research provided a new possibility for preventing and treating hyperlipidemia.

The present protocol describes an integrated strategy for exploring the key targets and mechanisms of Fructus Phyllanthi against hyperlipidemia based on network pharmacology prediction and metabolomics verification.

网络药理学预测及代谢组学验证 凤仙花抗高脂血症的机制

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug ...

使用 LC-MS 进行泪液蛋白质组学的非侵入性生物标志物发现

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

作者聚焦：使用蛋白质组学研究的标准化方案推进泪液分析 

This protocol describes a method for collecting tear samples using Schirmer strips and an integrated quantitative workflow for discovering non-invasive tear protein biomarkers. The suspension trapping sample preparation workflow enables fast and robust tear sample preparation and mass spectrometric analysis, resulting in higher peptide recovery yields and protein identification than standard in-solution procedures.

液相色谱-串联质谱法制备泪液蛋白质组学的蛋白质悬浮捕获样品

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for ...