Name: screening bioactive nanoparticles in phagocytic immune cells for inhibitors of toll-like receptor signaling
Uploaded: 2017-07-26T14:00:00
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...

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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<su...

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 &#176;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&#39;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...

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

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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Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

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High Content Screening in Neurodegenerative Diseases

在神经退行性疾病的高内涵筛选

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

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

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 ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

软琼脂集落形成实验利用识别的致瘤性乳腺癌细胞抑制剂

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...