Name: lucifer yellow - a robust paracellular permeability marker in a cell model of the human blood-brain barrier
Uploaded: 2019-08-19T13:00:00
Description: Watch this Scientific Journal Video about Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier at JoVE.com

作者感谢美国药学协会2017年新调查员奖、杜克斯内大学亨克勒恐惧病奖和Manickam实验室药学院启动基金的财政支持。我们要感谢泄漏实验室（杜克斯纳大学）的西方印迹援助，并允许使用他们的奥德赛16位成像仪。我们还要特别感谢坎达普·戴夫（Manickam实验室）对西方印迹的帮助。

1.一般 hCMEC/D3 细胞培养
<ol><li>冷冻细胞的复苏 注：所有细胞培养维护和实验均在无菌生物安全罩内进行。培养培养剂、补充剂和试剂要么作为无菌产品购买，要么通过过滤使用0.22 μm膜过滤器进行灭菌，以防止微生物污染。<ol>
<li>在组织培养瓶中加入8.5 mL的胶原蛋白溶液（0.15mg/mL）（75cm2生长面积;以后称为T75），并将其放入培养箱（37°C，5%CO2）1小时。</li>
		<li>取...

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BBB的一个关键作用是防止非必需离子和有毒物质在系统循环和大脑之间交换，以保持神经微环境的四分法。BBB 的一个特征是毛细管内皮细胞能够形成紧密的结（TJs），从而有效密封半细胞运输路线。我们演示了一种LY P应用测定作为定量方法，以确定培养hCMEC/D3单层中TJ阻隔形成表动力学。通过西方印迹正交检测的 ZO-1 表达式验证了 LY P应用研究中的数据，详于下一段所述。作为已?...

血脑屏障（BBB）是限制血浆成分流入脑组织的保护屏障，由脑内皮细胞和围细胞等辅助细胞组成。BBB的主要作用是作为屏障，密封外周血和中枢神经系统（CNS）之间的空间，以保持神经微环境1，2的血缘。脑毛细管内皮细胞通过形成细胞间紧密结（TJs）1有效密封准细胞通路。这种保护屏障允许葡萄糖和选定的营养物质进入大脑，同时防止大多数离子、有毒物质和药物通过这个紧密的屏障。除了保护作用外，BBB的天然屏障功能对开发针对CNS的药物输送系统提出了严峻的挑战。
BBB的体外细胞培养模型是研究其生物学和了解药物治疗对TJ屏障完整性影响的有用工具。我们使用人脑微血管内皮细胞系（hCMEC/D3）作为体外模型，因为它是人脑内皮3的公认模型，并重述了人类BBB的许多功能。hCMEC/D3是最常见的细胞系之一，用于在体外4、5、6、7、8、9对BBB进行建模。尽管这种细胞系的跨端电阻值相对较低，是阻隔性紧度的一个指标，但该细胞系保留了大脑内皮细胞的大部分形态和功能特性，即使在没有共培养的胶质细胞6，7。hCMEC/D3细胞系表示多个BBB标记，包括活性转运体和受体，直到大约通过35，而不经过分化到不稳定的表型6，7，9 ，10，11.hCMEC/D3细胞系作为体外BBB模型最显著的特征是能够形成TJs5，9，11，12。应该指出的是，虽然干细胞衍生的BBB模型在许多研究中表现出更高的渗透性，与hCMEC/D3细胞系相比，他们确实表达了一些BBB标记，但它们尚未演变为最常见的BBB细胞模型13。重要的是，干细胞衍生的BBB模型仍然具有最大通道数的特征，使细胞能够保持稳定的BBB表型14。
三种主要方法通常用于确定TJ阻隔完整性，包括TEER的测量，测量小亲水性示踪分子（P应用）的表观渗透系数（P应用），如蔗糖、黄素、路西法黄、等和免疫染色的已知分子标记的TJ，如克劳丁-5，ZO-1，卡他丁等5。TEER是一种相对简单和定量的方法，用于测量在多孔膜基板上培养的细胞单层的电阻。但是，TEER 值可能受实验变量（如培养介质的组成和测量仪器的类型）的影响。这些因素的可能组合导致在 hCMEC/D3 细胞系中广泛分布 TEER 值，范围从2 到 1150 Ω cm 2，培养2-21天13。免疫染色是一种视觉方法，通过使用抗体标记靶向蛋白来确定TJ蛋白的存在。然而，免疫染色涉及一系列实验步骤，包括需要修复/渗透可能导致实验伪影的细胞，荧光信号可能会随着时间的推移而消退。上述因素可能导致主观错误影响数据质量。
这项工作的主要重点是提出基于LY的表观渗透性测定，以确定培养的hCMEC/D3细胞中汇体单层形成的动力学。虽然其他先进的体外BBB系统，如共培养系统，微流体系统，是生理上更相关的模拟与显著改善屏障功能15，16，17，hCMEC/D3横井设置是一个简单可靠的模型，用于估计TJ形成的动力学，并快速筛选不同药物制剂对阻隔功能的影响。通常，P应用值对于 hCMEC/D3 单层中的各种亲水溶性溶质是一致的。例如，报告不同体外BBB模型中各种低分子质量溶质（如蔗糖、曼尼醇、LY等）的P应用值在10-4厘米/分钟5，18，19 ,在我们的实验设置中，大脑内皮细胞被播种在胶原蛋白涂层的微孔膜上，用于细胞附着和单层形成，以模仿体内屏障。在圆锥侧添加的LY预计将穿过细胞间紧密结，并在巴索侧累积。巴利侧LY浓度越大，表明存在不成熟、功能不全的屏障，而较低的浓度则反映了由于功能性TJs的存在导致成熟的屏障而导致运输受限。
LY 是一种亲水染料，具有明显的激发/发射峰值，无需放射性标签示踪分子，如蔗糖、曼尼醇或乌林。因此，LY的荧光值可用于直接计算其在BBB单层上的准细胞渗透性。此外，与许多用于生物医学领域的商业染料，遭受小斯托克斯变化，如荧光21，斯托克斯转移LY是约108nm有足够的光谱分离，从而允许LY荧光数据作为强健的读出，以确定副细胞渗透性。我们使用西方印迹作为正交技术来演示紧密结标记蛋白 ZO-1 在培养时间中表达的变化。通过西方印迹检测到的ZO-1表达式用于补充LY P应用数据，并结合这些数据表明，在LY P应用值中观察到的变化反映了单层的形成，并逐渐增加紧密交汇点标记 ZO-1 的表达式。
如前所述，这项工作的中心重点是演示LY测定作为一种简单的技术，以监测具有功能紧密结的交汇单层的形成。然而，为了证明所开发检测的另一个效用，我们测量了DNA纳米粒子转染hCMEC/D3单层中的LY P应用。核酸可以通过带正电荷的聚合物组与核酸的负电荷磷酸盐组之间的静电相互作用，浓缩成直径为100-200nm的聚电解质纳米颗粒。23.在我们的工作中，我们将这些复合物称为DNA纳米粒子（DNA NPs）。虽然我们的目的是转染细胞和表达所需的蛋白质，但我们必须确保 hCMEC/D3 单层的屏障特性不会受到损害。我们的数据表明，标准的4 h荧光酶基因转染机制不能显著改变LY渗透性，证明LY P应用测定的效用，以确定TJ屏障完整性的变化。

<table border="0" cellpadding="0" cellspacing="0" style="width:1035px;" width="1033">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">hCMEC/D3 cell line</td>
			<td style="width:195px;">Cedarlane Laboratories</td>
			<td style="width:123px;">102114.3C-P25</td>
			<td style="width:172px;">human cerebral microvascular endothelial cell line&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gWizLuc</td>
			<td>Aldevron&#160;</td>
			<td>5000-5001</td>
			<td>Plasmid DNA encoding luciferase gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lucifer yellow CH dilithium salt&#160;</td>
			<td>Invitrogen</td>
			<td>155267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transwell inserts with polyethylene terephthalate (PET) track-etched membranes</td>
			<td>Falcon</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture flask&#160;</td>
			<td>Olympus Plastics</td>
			<td>25-207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Flat Bottom&#160;</td>
			<td>Olympus Plastics</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Black 96-Well Immuno Plates&#160;</td>
			<td>Thermo Scientific</td>
			<td>437111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S-MEM 1X</td>
			<td>Gibco</td>
			<td>1951695</td>
			<td>Spinner-minimum essential medium (S-MEM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EBM-2</td>
			<td>Clonetics</td>
			<td>CC-3156</td>
			<td>Endothelial cell basal medium-2(EBM-2)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate-buffered saline 1X</td>
			<td>HyClone</td>
			<td>SH3025601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type I&#160;</td>
			<td>Discovery Labware, Inc.</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Pierce BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Culture Lysis 5X Reagent&#160;</td>
			<td>Promega</td>
			<td>E1531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beetle Luciferin, Potassium Salt&#160;</td>
			<td>Promega</td>
			<td>E1601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">SpectraMax i3&#160;</td>
			<td style="width:195px;">Molecular Devices</td>
			<td style="width:123px;"></td>
			<td>Fluorescence Plate Reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:195px;">Gibco</td>
			<td style="width:123px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">ZO-1 Polyclonal Antibody&#160;</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:123px;">61-7300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">anti-GAPDH antibody</td>
			<td style="width:195px;">abcam</td>
			<td style="width:123px;">ab8245</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:545px;">Alexa Fluor680-conjugated AffiniPure Donkey Anti-Mouse LgG(H+L)</td>
			<td style="width:195px;">Jackson ImmunoResearch Inc</td>
			<td style="width:123px;">128817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">12-well, Flat Bottom</td>
			<td style="width:195px;">Olympus Plastics</td>
			<td style="width:123px;">25-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">RIPA buffer (5X)</td>
			<td style="width:195px;">Alfa Aesar</td>
			<td style="width:123px;">J62524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Aprotinin</td>
			<td style="width:195px;">Fisher BioReagents</td>
			<td style="width:123px;">BP2503-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Odyssey CLx imager</td>
			<td style="width:195px;">LI-COR Biosciences</td>
			<td style="width:123px;"></td>
			<td>for scanning western blot membranes</td>
		</tr>
	</tbody>
</table>

lucifer yellow - a robust paracellular permeability marker in a cell model of the human blood-brain barrier

血脑屏障BBB由内皮细胞组成，在全身循环和大脑之间形成屏障，防止非必需离子和有毒物质的交换。紧密的结（TJ）有效地密封了单层中的准细胞空间，从而产生了完整的屏障。本研究描述了一种基于LY的荧光测定，可用于确定其表观渗透系数（P应用），进而可用于确定连接单层形成的动力学和由此产生的紧密结hCMEC/D3 单层的屏障完整性。我们进一步演示了此测定的另一个效用，以确定转染细胞中的TJ功能完整性。我们从LY P应用测定数据显示，在跨井设置中播种的hCMEC/D3细胞有效地限制了LY准细胞传输7天后培养。作为所呈现的测定的另一个效用，我们还证明了DNA纳米粒子转染不会改变hCMEC/D3单层中的LY准细胞传输。

首先，我们确定了培养时间对LY渗透性的影响，以确定TJ形成的表观动力学。从第 1 天到 10 天后种子的均值 LY P应用值如图2a所示。在第 1 天，平均 P应用为 4.25 x 10-4厘米/分钟，第 2 天略降至 3.32 x 10-4厘米/分钟。平均 P应用值在第 3 天略微增加到 3.93 x 10-4厘米/分钟，直到第 6 天才出现显著变化。与第 1 ...

Watch this Scientific Journal Video about Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier at JoVE.com

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

路西法黄 - 人类血脑屏障细胞模型中一个健壮的细胞渗透标记

我们提出了荧光测定，以证明Lucifer黄色（LY）是一个强大的标记，以确定hCMEC/D3细胞单层的明显准细胞渗透性，人类血脑屏障的体外模型。我们使用这种测定来确定培养的hCMEC/D3细胞中汇体单层形成的动力学。

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...

该协议是一种强大的技术，用于确定人类血脑屏障细胞模型中细胞间紧密结的功能完整性。这是一个简单且相当便宜的协议，允许在更短的时间段内计算明显的渗透性，而不是运行更长的动力学测定。该方法还可用于确定脑内皮细胞中与聚合物DNA纳米粒子转运的紧密结的功能完整性。要开始细胞电镀，请将带微孔膜的组织培养物插入 24 孔培养板。在每个组织培养物插入中加入 400 微升 0.15 毫克每毫升胶原蛋白类型 1。然后在37摄氏度、5%的二氧化碳孵化器中孵育一小时。轻轻摇动 24 孔板，使胶原蛋白溶液在组织培养物中的微孔膜上甚至扩散。一小时后，取出胶原蛋白溶液，用0.4毫升1X PBS缓冲液轻轻清洗微孔膜。现在，板hCMEC/D3细胞的密度为每平方厘米5万个细胞在细胞插入。将带有组织培养装置的 24 井板放在培养箱中，以允许细胞附着和增殖。孵育板7天，使细胞达到100%的汇合。每隔一天去除生长介质，将0.5毫升预热新鲜介质转移到组织培养剂中。重复西印迹的电镀过程，以确定DNA纳米粒子转染的ZO-1表达变化，以及ATP测定，以确定转染细胞中的细胞生存能力。为确定路西法黄的明显渗透性，在播种后的每一天，去除生长介质，并在井底板侧添加1.5毫升预热运输缓冲液。现在，在每个跨井插入的面添加 58.3 微升 20 微摩尔路西法黄色溶液。保存 50 微升溶液，用于荧光测量。在从一边完全去除残留的PBS缓冲液后，尽快加入路西法黄色溶液，以避免细胞干燥。确保在一边准确体积的路西法黄色溶液。确保所有刀片中添加相同且准确的路西法黄色溶液体积。快速工作以避免干燥细胞。在旋转板摇床中孵育细胞，温度为 100 RPM 和 37 摄氏度，持续 60 分钟。然后从每个阴面室中取出 30 微升路西法黄色样品。将 20 微摩尔路西法黄溶液和面样品转移到预标记管中。现在使用传输缓冲器稀释样品十倍。从每个基底器隔间中取出 500 微升，然后将样品转移到预标记的管中。为标准曲线准备一系列路西法黄色标准。将每个标准标准样品和基底样品的 100 微升添加到黑色 96 井板的每个井中。使用荧光微孔板读取器测量路西法黄色荧光强度并计算明显的渗透性，如手稿文本中所述。利用栽培时间对路西法黄渗透性的影响，确定紧密结的形成表观动力学。与第一天相比，第七天明显的渗透值显著下降，表明屏障变得更紧。然后，明显的渗透值稳定下来，直到第10天。这意味着屏障的形成是完整的和功能，导致路西法黄色副细胞传输减少。西印迹用于检测紧结蛋白ZO-1随时间变化。两个波段表示两个 ZO-1 等构形式。密度测定分析表明，ZO-1的像素值从播种后的第三天增加到第七天，表明紧密结点蛋白ZO-1从三天到第七天持续形成。在播种后的第7天钙消耗处理后，ZO-1的带子几乎无法检测，表明ZO-1在没有钙离子的情况下无法形成。如结果所示，可以进行ZO-1蛋白的西印，以确定紧密结点蛋白表达水平的变化，补充路西法黄为基础的功能活性数据。我们还发现，人脑内皮细胞中紧密结点的功能活性不受DNA纳米粒子转染的影响。

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

跨越大鼠血脑屏障FITC加载铁蛋白体外渗透：一个模型来研究Nanoformulated分子的交付

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

利用大分子挤出的人类食性增大疤痕的体外模型

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

实时活体内多光子显微镜可可视化聚焦超声和微气泡处理，以提高血脑屏障通透性

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

构建人主动脉平滑肌细胞器官芯片模型，用于概括主动脉壁中的生物力学应变

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model

在大鼠模型中使用低强度聚焦超声破坏血脊髓屏障

Low-intensity focused ultrasound (LIFU) uses ultrasonic pulsations at lower intensities than ultrasound and is being tested as a reversible and precise ...