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Neuroscience

通过跨内皮电阻检测对bEnd.3血管内皮细胞进行屏障功能完整性记录

Published: September 29, 2023
doi:
10.3791/65938
, , , , , , ,
1School of Pharmacy/School of Modern Chinese Medicine Industry,Chengdu University of Traditional Chinese Medicine, 2Meishan Hospital of Chengdu University of Traditional Chinese Medicine, 3School of Ethnic Medicine,Chengdu University of Traditional Chinese Medicine, 4School of Pharmacy,Ningxia Medical University, 5Innovative Institute of Chinese Medicine and Pharmacy/Academy for Interdiscipline,Chengdu University of Traditional Chinese Medicine

Summary

该协议描述了一种可靠且有效的脑血屏障 体外 模型。该方法使用小鼠脑血管内皮细胞bEnd.3并测量跨膜电阻。

Abstract

血脑屏障（BBB）是由微血管内皮细胞、星形胶质细胞和周细胞组成的动态生理结构。通过协调有害物质的受限转运、营养吸收和大脑中代谢物清除之间的相互作用，血脑屏障对于维持中枢神经系统稳态至关重要。建立血脑屏障的 体外 模型是探索神经系统疾病的病理生理学和创建药物治疗的宝贵工具。本研究描述了一种通过将 bEnd.3 细胞接种到 24 孔板的上腔室中来创建 体外 单层 BBB 细胞模型的过程。为了评估细胞屏障功能的完整性，采用常规的上皮细胞电压表实时记录正常细胞和CoCl2诱导的缺氧细胞的跨膜电阻。我们预计上述实验将为创建BBB的 体外 模型和治疗中枢神经系统疾病的药物提供有效的思路。

Introduction

血脑屏障是血液循环和神经组织之间独特的生物界面，由血管内皮细胞、周细胞、星形胶质细胞、神经元和其他细胞结构组成1。离子、化学物质和细胞在血液和大脑之间的流动受到该屏障的严格调节。这种稳态可以保护神经组织免受毒素和病原体的侵害，同时也使大脑神经能够正常运作2,3。维持血脑屏障的完整性可以有效预防影响中枢神经系统的疾病的发展和进展，如神经元功能障碍、水肿和神经炎症4.然而，BBB独特的生理特性阻止了超过98%的小分子药物和100%的大分子药物进入中枢神经系统5。因此，在中枢神经系统药物开发过程中增加药物通过血脑屏障的渗透对于实现治疗效果至关重要 6,7。尽管底物的计算机模拟筛选大大提高了候选药物穿越 BBB 的概率，但仍需要可靠且负担得起的体外/体内 BBB 模型来满足科学研究的需要8。

一种快速且经济实惠的高通量药物筛选技术是体外模型9。为了阐明药物对血脑屏障功能影响的基本过程及其在疾病发展和进展中的作用，已经创建了一系列简化的体外血脑屏障模型。目前，常见的体外BBB模型有单层、共培养、动态和微流控模型10,11,12，由血管内皮细胞和星形胶质细胞、周细胞或小胶质细胞13,14构建。尽管 3D 细胞培养物更符合 BBB15 的生理结构，但它们作为 BBB 药物筛选手段的应用仍然受到其复杂设计和低于标准的可重复性的限制。相比之下，单层体外模型是最常用于研究BBB的模型，适用于确定特定细胞中膜转运蛋白和紧密连接蛋白的表达。

跨膜电阻 （TEER） 测量是一种评估和监测电阻层的细胞层并评估屏障的细胞完整性和渗透性的技术。通过同时将两个电极插入单层两侧的生长培养基或缓冲溶液中，可以测量通过电池致密层16,17的交流电或电阻抗。为了确定体外BBB模型是否已正确创建，TEER的测量通常将采用作为金标准18。另一方面，通过测量药物受累后细胞层电阻的变化，可以准确预测药物作用对BBB通透性的趋势19。例如，Feng等人发现，地黄科的主要活性单体楸波尔（地黄科的主要活性单体）可以有效逆转脂多糖诱导的血脑屏障中紧密连接蛋白的下调，提高小鼠脑内皮细胞层20的TEER值。

神经炎症反应通常是血脑屏障稳态失衡的主要原因21.低氧治疗诱发神经炎症损伤是破坏血脑屏障的主要方法，主要包括物理方法和化学试剂方法。前者主要利用三气体培养箱来改变细胞生长环境中的氧含量以模拟缺氧条件22，而后者是通过人为地将脱氧试剂（如CoCl2）引入细胞培养基23来实现的。如果 Fe2+ 被血红素中的 Co2+ 取代，细胞将保持脱氧状态。如果催化基团中的Fe2+被Co2+取代，则脯氨酸羟化酶和天冬氨酸羟化酶活性将受到抑制，导致缺氧诱导因子-1α（HIF-1α）24的积累。在持续缺氧下，细胞质中HIF-1α的去磷酸化触发细胞死亡并激活血管内皮生长因子，最终提高血管通透性。在先前的研究 25,26 中，已经充分证明缺氧可以显着降低内皮紧密连接蛋白的表达，从而增加 BBB 的通透性。在这项研究中，测量了接种在 24 孔板中的 bEnd.3 细胞的时间电阻曲线，以创建一个简单的 BBB 模型。使用该模型，我们表征了 CoCl2 干预后细胞 TEER 的变化，以构建可用于筛选 BBB 保护药物的细胞模型。

Protocol

注：将小鼠脑源性内皮细胞.3 （bEnd.3） 接种到 24 孔板的腔室中，以在特定培养基条件下构建简单的 BBB 体外 模型。通过TEER仪测量正常细胞和缺氧细胞的TEER（图1 和 图2）。 1. 溶液制备 制备含有FBS（10%，v / v），100U / mL青霉素和10mg / mL链霉素的DMEM细胞培养基（参见 材料表）。 通过向…

Representative Results

该协议允许根据跨内皮电阻仪中设置的参数记录细胞电阻值的变化。采用CCK-8法筛选不同浓度CoCl2处理的bEnd.3细胞（活细胞数）。CoCl2 产生的细胞损伤越大，细胞活力越低。我们发现 300 μM 的 CoCl2 在体外具有显着的细胞毒性，该浓度用于后续实验（图 3A）。通过监测bEnd.3的生长抗性变化，我们发现细胞TEER值在第5天开始稳定。在第6 …

Discussion

大脑是最发达的身体器官之一，控制着各种复杂的生理过程，包括记忆、认知、听觉、嗅觉和运动27.大脑是人体最复杂、最患病的器官之一。由于空气污染、饮食习惯不规律和其他因素，许多中枢神经系统疾病的发生呈逐年增长趋势 27,28,29。有趣的是，一些中枢神经系统疾病的开始和进展，如中风、癫痫和阿…

Disclosures

The authors have nothing to disclose.

Acknowledgements

感谢国家自然科学基金（82274207 82104533）、宁夏重点研发计划（2023BEG02012）、成都中医药大学星林学者研究推进项目（XKTD2022013）的资助。

Materials

24-well transwell plate Corning (Corning 3470, 0.33 cm2, 0.4 µm) 10522023
75 % ethanol ChengDu Chron Chemicals Co,.Ltd 2023052901
96-well plate Guangzhou Jet Bio-Filtration Co., Ltd 220412-078-B
bEnd.3 cells Hunan Fenghui Biotechnology Co., Ltd CL0049
Cell counting kit-8 (CCK-8) Boster Biological Technology Co., Ltd BG0025
Cell culture dish (100mm) Zhejiang Sorfa Life Science Research Co., Ltd 1192022
Cobalt Chloride (CoCl2) Sigma 15862
DMSO Boster Biological Technology Co., Ltd PYG0040
Dulbecco's modified eagle medium (1x) Gibco ThermoFisher Scientific 8121587
Fetal bovine serum Gibco ThermoFisher Scientific 2166090RP
GraphPad Prism software GraphPad Software 9.0.0(121)
Matrigel (Contains collagen IV) MedChemexpress HY-K6002
Microplate reader Molecular Devices SpectraMax iD5
OriginPro 8 software OriginLab Corporation v8.0724(B724)
Penicillin-Streptomycin (100x) Boster Biological Technology Co., Ltd 17C18B16
Phosphate buffered saline (PBS, 1x) Gibco ThermoFisher Scientific 8120485
Sodium hypochlorite ChengDu Chron Chemicals Co,.Ltd 2022091501
Transmembrane resistance meter World Precision Instruments LLC VOM3 (verison 1.6)
Trypsin 0.25% (1x) HyClone J210045
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Tags

Keywords: Blood-brain BarrierBBB ModelBEnd.3 CellsTransendothelial Electrical Resistance (TEER)Barrier IntegrityCell PermeabilityIn Vitro ModelCentral Nervous SystemDrug ScreeningHypoxia
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Fan, F., Jiang, H., Hou, Y., Zhang, Y., Zhao, Q., Zeng, Y., Meng, X., Wang, X. Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection. J. Vis. Exp. (199), e65938, doi:10.3791/65938 (2023).
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