Name: screening bioactive nanoparticles in phagocytic immune cells for inhibitors of toll-like receptor signaling
Uploaded: 2017-07-26T14:00:00.000+00:00
Duration: 9 min 51 s
Description: Watch this Scientific Journal Video about Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling at JoVE.com

作者要感谢上海市第一人民医院（HY）起始基金，上海交通高峰临床医学奖学金支持，上海市高校特聘任（东方学者）教授的支持大学医学院（HY），以及加拿大克罗恩病和结肠炎基金会（CCFC）（SET和HY）的资助。

1.细胞培养基和试剂的制备<ol><li> 通过将10％胎牛血清（FBS），2mM L-谷氨酰胺和1mM丙酮酸钠的补充剂加入到RPMI-1640培养基中来制备完整的细胞培养基R10。 <ol><li>通过向R10加入抗生素Zeocin（200μg/ mL）来制备选择培养基R10-Z，以维持在NF-κB/ AP-1活化的控制下的SEAP表达。为了选择表达SEAP和荧光素酶报告基因的细胞，将ROC（100μg/ mL）和杀稻瘟素（10μg/ mL）添加到R10（作为R10-ZB）中。 </li></ol></li><li> 通过溶解在基板粉末的一个袋（E .G。，QUANTI -蓝）到100ml超纯，无内毒素的水在一个干净的125姆·格拉斯烧瓶中制备SEAP底物溶液。 <ol><li>轻轻旋转溶液，并在37℃下孵育1小时以确保底物完全溶解。 </li> &lt;li&gt;使用0.2μm的膜过滤溶液，以确保其无菌性（可选），并在使用前将其储存在4°C至多2周。 </li></ol></li><li>通过将一个底物粉末（ 例如 QUANTI-Luc）溶解在无菌的50mL离心管中的25mL超纯无内毒素游离水中来制备萤光素酶底物溶液。 <ol><li>完全溶解粉末后立即使用溶液。或者，使用前将溶液储存在4°C（最多一周）或-20°C（最多一个月）。 注意:两种基材溶液都是光敏的，每当有可能时应避免曝光。溶液的多次冻融循环可能导致底物的不稳定性并缩短其保质期。 </li></ol></li><li>在分子级二甲基亚砜（DMSO）中制备佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）的储备溶液，使其浓度为500μg/ mL。制备储备溶液的等分试样（在500μL管中10μL）并将其储存在-20℃。 </li><li>在无菌，无内毒素的水中以5 mg / mL的浓度制备LPS（大肠杆菌K12）储备溶液，并将等分试样长期储存在-20°C。通过将原料LPS稀释到浓度为100μg/ mL的磷酸盐缓冲盐水（PBS）中并在使用前储存在-20℃来制备工作的LPS溶液。 注意:培养用途的试剂制剂应在生物安全柜内进行，所有容器应在使用前进行灭菌。应避免PMA和LPS溶液的重复冻融循环。 </li></ol> 2.培养THP-1报道细胞来源的巨噬细胞<ol><li> 使用两个THP-1报道细胞系:THP-1-XBlue和THP-1-Dual细胞。前者具有由NF-κB/ AP-1控制的SEAP报告基因，后者具有双重报道基因系统，相同的SEAP报告基因和IRF控制的荧光素酶基因。该ir文化程序是一样的，除了选择培养基。 <ol><li>在10 mL R10培养基中解冻工作细胞储备（约5 x 10 6个细胞），以300 xg的速度将细胞转移5分钟。将细胞重悬于10mL的R10培养基中，并将其转移到T75培养瓶中。每2-3天更换培养基，直到细胞达到1×10 6个细胞/ mL的密度。 </li><li>当细胞生长达到其能力时，通过细胞以将细胞密度降低至2-5×10 5个细胞/ mL的范围，因此细胞可以继续生长。 </li><li>至少1次传代后，开始培养选择培养基中的细胞:THP-1-XBlue的R10-Z和THP-1-Dual的R10-ZB。在用选择培养基培养细胞至少1次后，细胞准备进行实验。 注意:细胞过度生长可导致明显的细胞死亡;细胞存活率应保持&gt; 98％。记录通道号作为细胞在许多段落（&gt; 20段）之后，ls可能会有所不同。 注意:细胞培养瓶可以多次重复使用以节省成本;然而，这种做法可能会增加不同烧瓶之间一般污染和交叉污染的风险（如果同时处理不同的细胞系）。在培养基更换和细胞通过过程中，离心分离设定为300×g，持续5分钟。 </li></ol></li><li> 为了进行报道细胞测定，将种子细胞置于96孔平底细胞培养板中并将其分化成巨噬细胞。以下对该步骤进行说明。 <ol><li>将细胞从烧瓶转移到离心管中，以300×g离心细胞5分钟，并以1×10 6个细胞/ mL的浓度将其重悬于R10培养基中。将PMA溶液的等分试样加入到终浓度为50ng / mL的细胞悬浮液中。 </li><li>将100μL细胞悬浮液转移到96孔的每个孔中使用多通道移液器盘。在37℃孵育平板（细胞培养箱中）24小时。 </li><li>孵育后，使用真空吸气器（或多通道移液管）小心地取出培养基，并用PBS（100μL/孔）轻轻洗涤细胞两次;在每个孔中加入100μL新鲜的R10培养基。在进行报告测定之前，在培养箱中将细胞静置2天。 注意:休息步骤对于允许细胞在PMA刺激后冷静到正常的安静状态非常重要。这可以显着降低报告基因在未刺激条件下的背景信号。 注意:用PMA进行24次刺激后，将细胞分化为巨噬细胞样表型，具有孔底部的细胞粘附特征，以及伪足的形态特征。 </li></ol></li></ol> 3.使用报道细胞筛选潜在的TLR4纳米抑制剂</p&gt; 注意:由于TLR4信号利用了依赖于MyD88和TRIF的途径，因此被选为涵盖广泛TLR信号通路的主要靶标。 THP-1-XBlue报告细胞主要用于检测NF-κB/ AP-1激活，而THP-1-Dual细胞用于从TRIF依赖信号转导中激活IRF。 <ol><li> 通过在筛选之前产生剂量 - 反应曲线来确定最佳的LPS剂量。 <ol><li>在R10培养基中稀释工作的LPS溶液至0.01-100ng / mL的终浓度（以log 10级稀释）。布置板设计，并在96孔圆底培养板中稀释。确保每个孔在稀释后至少有110μL的溶液。 </li><li>使用真空抽吸器轻轻从细胞培养板（2.2.3）中取出培养基。 </li><li>转移稀释板（3.1.1）中制备的含有R10培养基的LPS i根据样品的布局，对培养板进行培养。在37℃孵育平板（细胞培养箱中）24小时。 </li><li> 24小时后，小心地将上清液（80μL/孔）转移到新的96孔圆底板中。立即对这些溶液进行比色和/或荧光素酶发光测定，或在分析开发之前将其储存在4℃（几小时）或-20℃（天）。 注意:实验和数据分析时，板布局的设计应该简单明了。对于每个条件，应考虑2-4个重复。始终包括阴性对照组（LPS null）。推荐使用THP-1-XBlue细胞进行NF-κB/ AP-1激活，因为双重细胞在分化成巨噬细胞后具有较高的NF-κB/ AP-1活化背景。然而，使用Dual细胞报告NF-κB/ AP-1和IRF活化是可行的。 </li></ol></li><li> 开发颜色NF-κB/ AP-1活化的荧光素酶检测和IRF活化的荧光素酶发光测定。 <ol><li>为了评估NF-κB/ AP-1活化，将每个样品的20μL上清液转移到新的96孔平底培养板中;并向每个孔中加入180μL预热的SEAP底物溶液。将板在37℃下孵育1-2小时以使显色（粉色至深蓝色）。在平板阅读器上收集655 nm的吸收。 注意:孵育时间可以根据颜色发展而变化（30分钟到孵育过夜）。建议等到最暗颜色的光密度（OD）达到1以上，同时避免色彩饱和度（OD&gt; 3）。 </li><li>为了分析IRF活化，将每个样品的10μL上清液转移到96孔透明的平底白底板中。加入荧光素酶溶液（50μL），并立即收集发光w呃好 注意:强烈建议使用具有自动注入功能的发光板读取器，以确保从孔到阱以及从板到板的发光读数的一致性。如果手动注射底物溶液，请确保每个孔的孵育时间和阅读设置一致。 </li></ol></li><li> 对具有10ng / mL LPS刺激的多种肽-GNP杂交体进行筛选测定（基于3.1）。按照与3.2相同的程序进行报告化验。 <ol><li>使用离心方法将肽-GNP杂交体在R10培养基中浓缩至200nM。将20体积的混合溶液（10nM）以18,000xg离心30分钟，并小心地丢弃上清液。将杂交体（管底部）收集到管中，用PBS洗涤两次，并将杂交体重新悬浮在一个体积的R10培养基中。 </li><li>混合等体积的浓缩杂交和含有R10培养基的LPS（20ng / mL）分别具有100nM和10ng / mL的混合物和LPS的终浓度。 </li><li>从培养板（2.2.3）中取出培养基，并将100μL混合溶液加入每个孔（每个条件重复3次）;包括阴性对照（仅培养基）和LPS对照（10ng / mL LPS，不含杂交体）。 </li><li>在37℃孵育24小时后，将每个孔的培养基转移到管中，并以18,000xg，4℃离心管30分钟。将上清液（50-80μL/管）收集到96孔圆底板中，并按3.2所述进行报道分析。 注意:离心后丢弃上清液，不要搅动管底部的杂交体。 注意:步骤3.3.4中的离心对于从培养基中去除非内在纳米颗粒杂交体是非常重要的，并且可以避免混合物与共培养物的干扰半数/发光读数。这是因为金纳米颗粒可以根据其尺寸和聚集吸收宽波长的光。 </li></ol></li></ol> 4.验证潜在候选人的抑制作用注意:为了确认来自筛选的潜在候选者的抑制作用，采用两种方法。一个是以固定的杂交浓度（或以其他方式）检查兴奋剂（LPS）的剂​​量反应;另一个是通过免疫印迹直接观察对NF-κB/ AP-1和IRF3信号的抑制。 <ol><li>在方法一中，按照3.3中所述的相同方法，用100nM的铅杂交体和1ng / mL和10ng / mL的两种LPS浓度进行报道测定。包括一个不活动的混合（基于筛选结果）作为混合对照进行比较。 </li><li> 对于免疫印迹方法，请遵循标准e实验程序。 <ol><li>在R10培养基中培养THP-1细胞，将细胞接种到12孔培养板（2×10 6个细胞/孔）中，通过用50ng / mL PMA处理细胞24小时，然后静息分化成巨噬细胞2天。 </li><li>在细胞分化后，随着时间的推移（至多4小时）用具有/不具有杂交体（100nM）的10ng / mL LPS刺激细胞。在不同的时间点（0,5,15,30,60,120和240分钟），制备用于免疫印迹的细胞裂解物。包括一个不活动的混合作为控件。 </li><li>探测IκBα，磷酸化p65和磷酸化IRF3的信号，以检测铅杂交体对NF-κB和IRF3活化的抑制作用。探测β-肌动蛋白和总IRF3信号作为内部对照。 注意:信号转导通常发生得更快（几小时），然后报告酶SEAP和荧光素酶的表达（24小时）。强烈推荐也穿孔作为另一种验证方法，在24小时测试的杂交体的生存力测定。 </li></ol></li></ol> 5.评估TLR特异性注意:为了研究前导肽-GNP杂交体的TLR特异性，测试了其他TLR信号通路，包括TLR2，TLR3和TLR5。 TLR7,8和9被排除在外，因为THP-1衍生的巨噬细胞由于在巨噬细胞34中缺乏TLR7,8和9的表达而对这些TLR的刺激不能很好地反应。 <ol><li>在两种报道细胞衍生的巨噬细胞上测试TLR2（Pam3CSK4），TLR3（poly I:C）和TLR5（鞭毛蛋白）特异性配体的各种浓度（1ng / mL至25μg/ mL），以获得相同的最佳浓度3.1和3.2中的程序。 </li><li>用铅混合物（100nM）和每种TLR配体的混合物以5.1获得的浓度处理细胞，以评估铅的抑制特异性根据3.3中描述的实验程序。包括不活动的混合动力作为对照进行比较。 </li></ol>

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作者没有什么可以披露的。

由于TLR参与许多炎性疾病的发病机制，因此已经成为调节免疫反应和炎症反应的治疗靶点。然而，迄今为止，抑制TLR信号通路治疗药物的临床发展取得了有限的成果。其抑制TLR7和TLR9的抗疟疾药物羟氯喹在临床用途35,36。同样，只有化合物的有限数量的已经进展到临床试验，包括依立托仑，一个TLR4拮抗剂，其显示出对强效抑制作用LPS介导的临床前研究37,38炎症应答，表现在相位阳性结果的I / II期临床试验39，40，41，但最终的III期临床试验未能降低死亡率的严重脓毒症患者42例 。这种失败有许多可能的原因，一个是脓毒症相关的炎症反应通常通过多个TLR途径触发，因此仅阻断TLR4可能不足以减少压倒性的炎症。因此，开发新型有效的多TLR抑制剂可以克服这些临床挑战，成为下一代抗炎治疗药物。预期这里描述的筛选策略和方案将成为研究TLR信号的有效实验工具，更重要的是作为加速搜索下一代TLR抑制剂的药物发现平台。 这种筛选方法在寻找新的TLR抑制剂方面提供了几个优点。首先，可以使用快速，敏感和定量的报道细胞系统以高通量方式实现筛选。其次，与报道细胞系，可以进行筛选以覆盖广泛的TLR信号级联，包括NF-κB/ AP-1途径和I型干扰素信号传导（通过IRF）;因此，它们是筛选多种TLR途径的理想选择。第三，这些报道细胞是从人单核细胞THP-1细胞系进行遗传工程改造的，这是一种很好的模型系统，用于研究针对先天免疫反应的干预措施。第四，容易维持细胞系，特别是可以分化成巨噬细胞;并且由于巨噬细胞在许多疾病相关的炎症病症中发挥关键作用，因此它们作为靶向TLR信号传导的免疫治疗药物的筛选的理想靶标。第五，单核细胞和巨噬细胞具有令人印象深刻的吞噬能力，​​其允许纳米颗粒的高细胞摄取，使得它们特别适用于研究纳米级治疗剂。此外，该筛选方案可以应用于不仅搜索基于纳米的方法中毒剂，还有其他类型的生物活性化合物。 虽然这个筛选平台是非常通用和健壮的，但是必须谨慎使用以避免发现错误。筛选主要基于报告基因测定，报告基因检测依赖于特定信号传导事件控制下报告基因的表达。理想情况下，报告基因表达（SEAP和荧光素酶）与感兴趣的信号转导途径的强度成正比，药物候选物的影响反映在测定读数中。然而，实际上，报告基因表达上游发生的任何生物事件都可能影响结果，有时会导致假阳性发现。例如，SEAP的低表达可能来自蛋白质合成过程的抑制，而不是TLR信号的下调43 。为了避免这种错误的发现，该标识的抑制活性d候选人应始终进行验证，黄金标准方法是通过免疫印迹直接观察信号通路。筛选纳米颗粒治疗剂的另一个重要方面是这些纳米器件的表面性质。基于表面改性剂，纳米装置可以具有各种生物活性。然而，它们也可能对某些生物分子具有非特异性结合能力。在非特异性结合SEAP或荧光素酶的情况下，对这些纳米器件的底物的催化活性可能受到影响，导致潜在的假发现。在筛选中包括额外的控制组（ 例如 ，仅限纳米设备）会降低发现错误的风险。最后但并非最不重要的是，必须检查鉴定的候选人的细胞毒性，以排除细胞毒性作为假发现的一个因素。这可以在筛选过程中同时使用标准生存力测定（ 例如 ，MTS或MTT），或在单独的实验中。

Toll样受体（TLR）是有助于抵御感染的第一道防线的先天免疫系统的关键要素之一。 TLR是负责通过信号转导1,2的级联通过识别的病原体相关分子模式（或的PAMP）全集感测侵入的病原体和安装防御反应。确定了10个人类TLR;除了其中配体保持不清楚的TLR10之外，每个TLR可以识别出不同的保守组的PAMP。例如，主要位于细胞表面的TLR2和TLR4可以分别从革兰氏阳性和革兰氏阴性菌中检测脂蛋白和糖脂;而主要存在于内体区的TLR3，TLR7 / 8和TLR9可以感染来自病毒和细菌的RNA和DNA产物3 。当被PAMP刺激时，TLR通过释放pro-inf来触发必需的免疫应答炎症介质，募集和激活效应免疫细胞，协调随后的适应性免疫事件4 。 TLR信号转导可被简单地分类为两个主要途径5,6。一种依赖于衔接蛋白骨髓分化因子88（MyD88） - MyD88依赖性途径。 TLR3以外的所有TLR均利用该途径激活活化的B细胞（NF-κB）和丝裂原相关蛋白激酶（MAPK）的核因子κ-轻链增强子，导致促炎介质如TNF- α，IL-6和IL-8。第二个途径利用含有TIR结构域的转录因子诱导型干扰素-β（TRIF） - TRIF依赖性或MyD88非依赖性途径来激活干扰素（IFN）调节因子（IRF）和NF-κB，导致产生I型IFN。完整的TLR信号对我们日常保护免受微生物和病毒感染至关重要; TLR信号通路缺陷会导致免疫缺陷，并且往往对人体健康有害。 7 然而，TLR信号是"双刃剑"，过度的，不受控制的TLR激活是有害的。过度活跃的TLR应答有助于发病机制在许多急性和慢性人类炎性疾病8,9。例如，败血症其特点是全身炎症反应和多器官损伤，主要是由于急性，铺天盖地的免疫反应对人感染，TLR2和TLR4在脓毒症病理生理学10，11，12发挥着至关重要的作用。此外，TLR5已被发现有助于囊性纤维化患者的慢性肺部炎症13， 14 。此外，失调的内体TLR信号传导（例如，TLR7和TLR9）强烈与若干自身免疫疾病包括全身性红斑狼疮（SLE）和类风湿性关节炎（RA）15，16的发展和进展有关。这些证据会聚线识别TLR信号作为许多炎性疾病17潜在的治疗靶。 尽管TLR应答的药理学调节预计是许多炎性病症是有益的，不幸的是，目前有可用的临床抑制TLR信号9，17，18非常少的化合物。这部分是由于涉及免疫稳态和疾病病理学的TLR途径的复杂性和冗余性。因此，寻找小说，可爱靶向多种TLR信号通路的治疗剂可以弥补基本差距，克服将TLR抑制剂推进临床的挑战。 在纳米科学和纳米技术的快速发展的光，纳米器件正在成为下一代由于其独特的性能19，20，23 TLR调节剂。纳米级尺寸允许这些纳米疗法有更好的生物分布和持续循环24，25，26。它们可以进一步官能化，以满足希望的药效学和药代动力学特性27，28，29。更令人兴奋的是，这些新型纳米器件的生物活性来自于它们的固有特性，可以为其定制具体的医疗应用，而不是简单地用作治疗剂的递送载体。例如，高密度脂蛋白（HDL） -样纳米颗粒被设计为通过清除所述TLR4配LPS 23抑制TLR4信号传导。此外，我们已经开发出一种肽-金纳米颗粒混合动力系统，其中，所述装饰的肽可以改变金纳米颗粒的表面特性，并允许它们具有各种生物活性30，31，32，33。这使得它们成为下一代纳米治疗剂的特殊类药物（或"纳米药物"）。 在这个方案中，我们提出了一种方法，以鉴定一类新型的肽 - 金纳米颗粒（肽-GNP）杂合体，其可以有效抑制吞噬免疫细胞中的多种TLR信号通路32 ， </sup&gt; 33 。该方法基于商业上可获得的THP-1报道细胞系。报道细胞由两个稳定的诱导型报道构建体组成:一个携带分泌的胚胎碱性磷酸酶（SEAP）基因，在由转录因子NF-κB和激活蛋白1（AP-1）诱导的启动子的控制下;另一种含有由干扰素调节因子（IRF）诱导的启动子控制下的分泌的萤光素酶报告基因。在TLR刺激下，信号转导导致NF-κB/ AP-1和/或IRFs的激活，其导致报道基因分泌SEAP和/或荧光素酶;可以使用分光光度计或发光计使用其相应的底物试剂容易地检测这些事件。使用这种方法筛选我们以前建立的肽-GNP杂交体的文库，我们确定了能够有效抑制TLR4信号通路的潜在候选物。铅肽 -然后使用另一种免疫印迹生物化学方法验证GNP杂交，并对其他TLR途径进行评估。这种方法可以快速，有效地筛选靶向TLR信号通路的新药。

<table><tbody><tr><td>THP-1-XBlue reporter cell</td><td>InvivoGen</td><td>thpx-sp</td><td>keep cell culture passage under 20</td></tr><tr><td>THP-1-Dual repoter cell</td><td>InvivoGen</td><td>thpd-nfis</td><td>keep cell culture passage under 20</td></tr><tr><td>RPMI-1640 (no L-glutamine)</td><td>GE Health Care</td><td>SH30096.02</td><td>Warm up to 37 &amp;deg;C before use; add supplements to make a complete medium R10</td></tr><tr><td>Fetal bovine serum (qualified)</td><td>Thermo Fisher Scientific</td><td>12484028</td><td>Heat inactivated; 10% in RPMI-1640</td></tr><tr><td>L-glutamine</td><td>Thermo Fisher Scientific</td><td>SH30034.02</td><td>2 mM in the complete medium R10</td></tr><tr><td>Sodium pyruvate</td><td>Thermo Fisher Scientific</td><td>11360-070</td><td>1 mM in the complete medium R10</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline, 1X, without calcium, magnesium</td><td>GE Health Care</td><td>SH30028.02</td><td>Use for cell washing and reagent preparation</td></tr><tr><td>QUANTI-Blue</td><td>InvivoGen</td><td>rep-qb1</td><td>SEAP substrate</td></tr><tr><td>QUANTI-Luc</td><td>InvivoGen</td><td>rep-qlc2</td><td>Luciferase substrate</td></tr><tr><td>Zeocin</td><td>InvivoGen</td><td>ant-zn-1</td><td>Selection antibiotics for reporter cells</td></tr><tr><td>Blasticidin</td><td>InvivoGen</td><td>anti-bl-1</td><td>Selection antibiotics for reporter cells</td></tr><tr><td>Dimethyl sulfoxide (DMSO) for molecular biology</td><td>Sigmal-Aldrich</td><td>D8418-100ML</td><td>Use for reagent preparation</td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA) for molecular biology</td><td>Sigmal-Aldrich</td><td>P1585-1MG</td><td>Use for cell differentiation</td></tr><tr><td>Lipopolysaccharide (LPS) from E. coli K12</td><td>InvivoGen</td><td>tlrl-eklps</td><td>TLR4 ligand</td></tr><tr><td>Pam3CSK4</td><td>InvivoGen</td><td>tlrl-pms</td><td>TLR2/1 ligand</td></tr><tr><td>Poly (I:C) HMW</td><td>InvivoGen</td><td>tlrl-pic</td><td>TLR3 ligand</td></tr><tr><td>Flagellin from S. Typhimurium (FLA-ST), ultrapure</td><td>InvivoGen</td><td>tlrl-epstfla</td><td>TLR5 ligand</td></tr><tr><td>SpectraMax Plus 384 microplate reader</td><td>Molecular Devices</td><td>N/A</td><td>Read colorimetric assay</td></tr><tr><td>Infinite M200 Pro multimode microplate reader with injectors</td><td>Tecan</td><td>N/A</td><td>Read luminiscience</td></tr><tr><td>Microfuge 22R centrifuge</td><td>Beckman Coulter</td><td>N/A</td><td>Temperature controlled micro-centrifugator (up to 18,000 g)</td></tr><tr><td>Allegra X-15R centrifuge</td><td>Beckman Coulter</td><td>N/A</td><td>Temperature controlled general purpose centrifugator (for cell culture use)</td></tr><tr><td>Costar assay plate, 96-well white with clear flat bottom, tissue culure treated</td><td>Corning Costar</td><td>3903</td><td>Used for luminiscence assay</td></tr></tbody></table>

screening bioactive nanoparticles in phagocytic immune cells for inhibitors of toll-like receptor signaling

Toll样受体（TLR）反应的药理学调节在许多炎性疾病的治疗中具有很大的前景。然而，迄今为止，已有有限的化合物可以减弱TLR信号，临床上没有临床批准的TLR抑制剂（抗疟药物羟氯喹除外）。鉴于纳米技术的快速发展，使用纳米器件的免疫反应性的操作可能为治疗这些疾病提供了新的策略。在这里，我们提出了一种高通量筛选方法，用于快速鉴定抑制吞噬免疫细胞中TLR信号传导的新型生物活性纳米颗粒。该筛选平台建立在具有比色和荧光素酶测定法的基于THP-1细胞的报道细胞上。通过两个诱导型报道构建体的稳定整合，报道细胞由人THP-1单核细胞系工程化。一个表达分泌的胚胎碱性磷酸酶（SEAP）基因在由转录因子NF-κB和AP-1诱导的启动子的控制下，另一种在由干扰素调节因子（IRF）诱导的启动子的控制下表达分泌的荧光素酶报道基因，TLR刺激后，报告细胞激活转录因子并随后产生SEAP和/或荧光素酶，其可以使用它们相应的底物试剂检测。使用我们以前研究中建立的肽 - 金纳米颗粒（GNP）杂交库作为例子，我们确定了一种能够有效抑制由其原型配体脂多糖（LPS）触发的TLR4信号级联两臂的肽-GNP杂交体。研究结果通过包括免疫印迹在内的标准生物化学技术进行验证。进一步的分析确定，这种铅杂交具有广泛的抑制谱，作用于多个TLR途径，包括TLR2,3,4和5.该实验方法允许快速评估ra纳米颗粒（或其他治疗化合物）可以调节吞噬免疫细胞中的特异性TLR信号。

整体实验方法如图1所示 。通过探测NF-κB/ AP-1和IRF的激活，两个THP-1报道细胞系THP-1-XBlue和THP-1-Dual分别用于快速筛选TLR反应。可以通过SEAP比色法检测NF-κB/ AP-1的活化，而通过萤光素酶发光监测IRF的活化。单核细胞THP-1细胞可以容易地衍生到巨噬细胞中，以筛选纳米装置对其在先天吞噬细胞免疫细胞上的免疫调节活性。记者系统可以高通量的方式进行筛选;这种方法对于发现新的免疫治疗剂是特别有用的，特别是针对先天免疫信号如TLR信号传导。 筛选程序和代表性结果如图所示图2A 和2B ）。首先测试不同浓度的TLR配体（TLR4作为实例），以获得肽-GNP杂交体的实际筛选的最佳浓度。 LPS引起的TLR4刺激导致NF-κB/ AP-1的活化和SEAP的产生。释放的SEAP转化了底物并改变了其光物理性质，这可以通过光吸收的变化来监测，导致溶液颜色变化（ 图2C ）。这种变化与刺激后释放的SEAP的量成比例，并且可以通过在分光光度计上测量655nm处的吸光度来定量（ 图2D ）。类似地，IRFs的激活（由LPS引发）导致luciferas的表达e，其催化底物产生发光（ 图2E ）。基于这些剂量反应，使用LPS（10ng / mL）的最佳浓度来筛选小型先前建立的肽-GNP杂交体的文库（ 表1 ）。该杂交种及其物理化学特性的制造在我们以前的出版物30，31，32进行了描述。从筛选中，鉴定出一组杂交体（P12及其衍生物）对由LPS引发的NF-κB/ AP-1和IRF活化的有效抑制活性（ 图2F ）;有趣的是，与肽包膜中的P12略微不同的杂合子P13没有任何抑制活性，可以作为混合对照进行比较。 P13衍生物显示出各种程度的轻度抑制活性，取决于其它r表面上装饰肽。 鉴定铅杂种后，重要的是验证抑制活性。首先通过检查杂种与LPS的不同比例来排除由于技术假象引起的潜在的假阳性结果来确认抑制。随着LPS浓度的增加，混合物（固定浓度）的抑制作用如预期那样降低（ 图3A） 和3B ）。为了进一步确保观察到的来自报道测定的抑制作用确实是通过引导杂交体下调NF-κB和IRF信号传导的结果，进行免疫印迹以随时间直接评估蛋白质信号转导。通过探测NF-κB亚基p65的磷酸化和NF-κB抑制剂IκBα的降解以及荧光体的荧光体来检测NF-κB和IRF3的活化分别为IRF3。 如图3C所示，引物杂合物P12可以减少p65磷酸化，抑制IκBα降解和延迟IRF3磷酸化，而无活性杂合P13不能（数据未显示）。所有这些结果证实，鉴定的铅杂交能够通过下调NF-κB和IRF3激活来抑制LPS介导的TLR4信号传导。 除TLR4信号外，还对TLR，TLR3和TLR5等其他TLR途径进一步评估了铅杂交体的抑制活性，以解决TLR特异性。 如图4所示，先导杂交P12能够减少TLR2-和TLR5介导的NF-κB/ AP-1信号传导以及TLR3介导的IRF活化;再次，无活性杂合体P13没有显示任何抑制活性。这些结果表明，鉴定的铅杂种具有很强的抑制作用<html>多个TLR途径的活动。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/56075/56075fig1.jpg" /> 图1:使用报道细胞测定的TLR抑制剂高通量筛选的总体实验方法。使用两种报道细胞系:THP-1-XBlue和THP-1-Dual。前者具有在NF-κB/ AP-1激活控制下的SEAP报告基因，而Dual系统在IRFs活化的控制下具有另外的荧光素酶报道基因。这些细胞可以容易地分化成巨噬细胞，用于免疫调节纳米颗粒在先天免疫信号上的高通量筛选。 <a href="//ecsource.jove.com/files/ftp_upload/56075/56075fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> / ftp_upload / 56075 / 56075fig2.jpg"/&gt; 图2:高通量筛选TLR4信号传导的纳米抑制剂。 （ A ）实验程序方案。 （ B ）分化巨噬细胞的光学显微镜图像（放大200倍）。 （ C ）来自SEAP报道分析的溶液颜色变化的代表性图像;根据培养基中的SEAP表达，底物溶液的颜色变成紫色或深蓝色（从原始粉红色）。 （ D ）响应于LPS刺激，在655nm处的SEAP底物吸收的定量分析。 （ E ）来自IRF报道系统的荧光素酶发光与LPS刺激成比例。 （ F ）高通量筛选，鉴定前导肽-GNP杂合物P12及其衍生物抑制TLR4信号传导的NF-κB/ AP-1和IRF途径;杂交浓度= 100nM; LPS浓度= 10 ng / mL;该条代表平均值±标准偏差; n = 2. <a href="//ecsource.jove.com/files/ftp_upload/56075/56075fig2large.jpg" target="_blank">请点击这里查看该图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/56075/56075fig3.jpg" /> 图3:铅混合抑制活性的验证。确认铅混合物与各种浓度的LPS对（ A ）SEAP和（ B ）荧光素酶报告系统的抑制作用。 （ C ）通过免疫印迹确认铅杂合物对NF-κB和IRF3信号传导的抑制作用。将非活性杂合体P13用作混合对照进行比较。杂交浓度= 100nM; LPS浓度= 10 ng / mL;该条代表平均值±标准偏差; n = 3; *和***表示p &lt;0.05 and p &lt;0.001，使用单因素方差分析。 <a href="//ecsource.jove.com/files/ftp_upload/56075/56075fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/56075/56075fig4.jpg" /> 图4:铅混合物对其他TLR信号通路的抑制作用。 TLR2刺激（Pam3CSK4 = 1 ng / mL）（ A ）和TL5刺激（鞭毛蛋白= 100ng / mL）（ B ）后，铅混合物对NF-κB/ AP-1的抑制作用。 （ C ）通过TLR3刺激后的铅杂合物（poly I:C =25μg/ mL），NF-κB/ AP-1和IRF信号传导的减少。杂交浓度= 100nM;该条代表平均值±标准偏差; n = 3; *，**和***分别表示p &lt;0.05，p &lt;0.01和p &lt;0.001单因素方差分析; ns:不重要。 <a href="//ecsource.jove.com/files/ftp_upload/56075/56075fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="表格1" src="/files/ftp_upload/56075/56075table1.jpg" /> 表1:我们早期研究中建立的一个小型肽-GNP杂交文库。 杂交体由金纳米颗粒核心（直径约13nm）和表面上的各种六肽涂层制成。 <a href="//ecsource.jove.com/files/ftp_upload/56075/56075table1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling at JoVE.com

筛选吞噬免疫细胞中的生物活性纳米颗粒以寻找 Toll 样受体信号传导抑制剂医学|视频

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Toll样受体（TLR）信号在许多人类炎性疾病的病理生理学中起着重要作用，并且预期在许多炎症状态下调节生物活性纳米颗粒的TLR应答是有益的。基于THP-1细胞的报道细胞提供了一个通用和强大的筛选平台，用于鉴定TLR信号传导的新型抑制剂。

筛选生物活性纳米颗粒在噬菌体免疫细胞中的Toll样受体信号传导抑制剂

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

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12.3K 次观看.  Shanghai Jiaotong University School of Medicine.  Toll样受体（TLR）信号在许多人类炎性疾病的病理生理学中起着重要作用，并且预期在许多炎症状态下调节生物活性纳米颗粒的TLR应答是有益的。基于THP-1细胞的报道细胞提供了一个通用和强大的筛选平台，用于鉴定TLR信号传导的新型抑制剂。 

视频: Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

在调节Toll样受体激活Neu1唾液酸酶活动的检测

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

科研

免疫与感染

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

生物工程

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

聚酐IL-1α纳米颗粒 体内抗肿瘤 活性评价

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

癌症研究

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

小胶质细胞作为替代生物传感器确定纳米颗粒神经毒性

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

神经科学

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

巨噬细胞报告器细胞分析，以检查聚合物表面的吸附蛋白层上的收费类似受体介导NF-kB/AP-1信号

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

化学抑制剂库筛选 t 细胞受体信号的中介识别

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

筛选生物活性纳米颗粒在噬菌体免疫细胞中的Toll样受体信号传导抑制剂

摘要

探索更多视频

此视频中的章节

在调节Toll样受体激活Neu1唾液酸酶活动的检测

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

聚酐IL-1α纳米颗粒体内抗肿瘤活性评价

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

小胶质细胞作为替代生物传感器确定纳米颗粒神经毒性

巨噬细胞报告器细胞分析，以检查聚合物表面的吸附蛋白层上的收费类似受体介导NF-kB/AP-1信号

化学抑制剂库筛选 t 细胞受体信号的中介识别

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

筛选生物活性纳米颗粒在噬菌体免疫细胞中的Toll样受体信号传导抑制剂

摘要

探索更多视频

此视频中的章节

在调节Toll样受体激活Neu1唾液酸酶活动的检测

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

聚酐IL-1α纳米颗粒 体内抗肿瘤 活性评价

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

小胶质细胞作为替代生物传感器确定纳米颗粒神经毒性

巨噬细胞报告器细胞分析，以检查聚合物表面的吸附蛋白层上的收费类似受体介导NF-kB/AP-1信号

化学抑制剂库筛选 t 细胞受体信号的中介识别

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

聚酐IL-1α纳米颗粒体内抗肿瘤活性评价