评估血脑屏障网状血管形成和破坏的体外测定
Summary
维持血脑屏障覆盖是中枢神经系统稳态的关键。该方案描述体外技术来描绘调节血脑屏障覆盖的基础和病理过程。
Abstract
血脑屏障（BBB）覆盖在中枢神经系统（CNS）的体内平衡中起着核心作用。 BBB由星形胶质细胞，周细胞和脑内皮细胞（BEC）动态维持。在这里，我们详细介绍使用永生化人类BEC的单一培养物，原代小鼠BEC的单一培养物和BBB的人源化三联培养模型（BEC，星形胶质细胞和周细胞）评估BBB覆盖率的方法。为了突出测定对疾病状态的适用性，我们描述了低聚淀粉样蛋白β（oAβ）对阿尔茨海默病（AD）进展的重要影响，对BBB覆盖的影响。此外，我们利用表皮生长因子（EGF）来阐明该技术的药物筛选潜力。我们的研究结果表明，单一和三重培养的BEC在基础条件下形成网状结构，并且oAβ破坏了这种细胞网状结构，并使预成型的网状结构退化，但EGF阻止了这种中断。因此，描述的技术对于解剖调节BBB覆盖的基础和疾病相关过程是重要的。
Introduction
大脑毛细血管的血脑屏障（BBB）是血液与脑接触的最大界面，在中枢神经系统（CNS） 1,2的稳态中起着核心作用。 BBB的动态过程阻止从血液中吸收不需要的分子，从CNS中除去废物，向CNS提供必需的营养物质和信号分子，并调节神经炎症1,2,3,4,5 。 BBB损伤在老化过程中是普遍存在的，并且包括阿尔茨海默病（AD），多发性硬化和中风1，2，3，4，5 ，ass =“xref”> 6。因此，BBB功能障碍可能在神经变性疾病中起关键作用，包括作为治疗靶点。
保持血管覆盖对于BBB的稳态功能是重要的。然而， 在体内和体外数据上是否涉及神经退行性疾病的过程引起更高或更低的BBB覆盖度6,7,8,9,10,11,12,13，特别是在AD中。因此，使用相关细胞类型的体外模型的发展有很强的理由来评估和更全面地了解BBB覆盖的动态。脑毛细血管由星形胶质细胞，周细胞和脑内皮细胞（BECs）组成， <sup class =“xref”> 3。所有细胞类型通过结构支持和通过分泌效应分子（如血管生成生长因子，细胞因子和趋化因子）有助于BBB的功能，其作用在旁分泌和自分泌样的方式。然而，BBB的主效应细胞是BEC3。通常，用于评估BBB功能的细胞培养技术是在加入应激物14,15,16之后对在滤器插入物上生长的细胞进行渗透性测定，或评估关键BEC蛋白的水平。虽然重要的是，这些检测不集中在脑血管覆盖。
这里，我们以前的方法17是详细的，以评估使用永生化人类BEC的单一培养物，原代小鼠BEC的单一培养物和人源化三重培养物的BEC覆盖和网状结构BBB的模型（BECs，星形胶质细胞和周细胞）。目标是证明对于BEC覆盖，被认为是AD进展的重要因素的oAβ的有害作用。表皮生长因子（EGF）的保护作用突出了该技术作为治疗筛选工具的潜力。该技术对于基础和应用研究具有几个广泛的应用，包括：1）描述特定途径对血管生成和血管覆盖的作用，2）评估疾病和衰老相关因素对血管生成和血管覆盖的影响，以及3）鉴定药理学目标。
Protocol
所有实验均遵循伊利诺斯大学芝加哥体育动物护理和使用委员会协议。 一般准备注意：脑微血管内皮细胞系（hCMEC / D3）是广泛表达的永生化人类BEC细胞系14,15,16,18,19。在基底内皮生长基础培养基中培养hCMEC / D3细胞在包被I型胶原（小牛皮，Hank的含有Ca 2+和Mg 2+的 Hank平衡盐溶液（HBSS）中的0.1％溶液1:20稀释）的组织培养瓶上培养（EBM-2），每5…
Representative Results
在单个培养物中，hCMEC / D3细胞（ 图3A ）和主要小鼠BEC（ 图3B ）都在整个孔中形成网状结构。结构的特点是互连节点的网格（ 图3 ）。在所有描述的范例（ 图1 ）中，网状结构在对照组中24小时后相似，形成约20个网状结构，总细胞长度约为10,000个像素。 <p class="jove_content" fo:keep-to…
Discussion
所描述的方法可用于解决围绕脑血管覆盖的几个基本生物学问题24 。具体来说，他们可以确定哪些受体和信号通路在血管发生，癌症组织中的血管覆盖以及与脑相关的外周内皮细胞中起作用。实例包括血管生成生长因子受体，一氧化氮，丝裂原活化蛋白激酶信号和钙信号传导25,26,27 。 hCMEC / D3和主要BECs可修改为遗传敲打方法，以促进这一努力18</sup…
Declarações
The authors have nothing to disclose.
Acknowledgements
莱昂泰由芝加哥伊利诺伊大学创业基金资助。
Materials
| hCMEC/D3 cells | Milipore | SCC066 | |
| EBM-2 basal media | Lonza | CC-3156 | |
| Collagen Type 1 | ThermoFisher | A1064401 | |
| HBSS, calcium, magnesium, no phenol red | ThermoFisher | 14025092 | |
| HBSS, no calcium, no magnesium, no phenol red | ThermoFisher | 14175095 | |
| Trypsin-EDTA (0.25%) | ThermoFisher | 25200056 | |
| Final concentrations of the SingleQuot growth factor supplements for EBM2 media | Lonza | CC-4147 | |
| 5% FBS | Lonza | CC-4147 | |
| 10% Ascorbic acid | Lonza | CC-4147 | |
| 10% Gentamycin sulphate | Lonza | CC-4147 | |
| 25% Hydrocortisone | Lonza | CC-4147 | |
| 1/4 volume of the supplied growth factors: fibroblast growth factor, epidermal growth factor, insulin-like growth factor, vascular endothelial growth factor | Lonza | CC-4147 | |
| Puromycin hydrochloride | VWR | 80503-312 | |
| MEM-HEPES | Thermo Scientific | 12360-038 | |
| Papain cell dissociation system (papain and DNase1) | Worthington Biochemical | LK003150 | |
| Human pericytes | Sciencell | 1200 | |
| Pericyte basal media | Sciencell | 1201 | |
| Pericyte growth supplement | Sciencell | 1252 | |
| Human Astrocytes | Sciencell | 1800 | |
| Astrocyte media | Sciencell | 1801 | |
| Astrocyte growth supplement | Sciencell | 1852 | |
| Basement membrane (Matrigel Growth Factor Reduced) | Corning | 356231 | |
| Angiogenesis m-plates (96-well) | ibidi | 89646 | |
| Human Epidermal growth factor | Shenendoah Biotechnology | 100-26 | |
| CellTracker green | ThermoFisher | C7025 | |
| CellTracker orange | ThermoFisher | C34551 | |
| CellTracker blue | ThermoFisher | C2110 | |
| Poly-l-lysine | Sciencell | 0403 | |
| 10% Neutral Buffered Formalin | Sigma-Aldrich | HT5012-60ML | |
| C57BL mice | Jackson Laboratory | na | |
| PCR tube strips | GeneMate | T-3014-2 | |
| Zeiss stereo discover v.8 dissecting microscope | Zeiss | na |
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Citar este artigo
Thomas, R., Diaz, K., Koster, K. P., Tai, L. M. In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption. J. Vis. Exp. (124), e55846, doi:10.3791/55846 (2017).