内皮细胞转吞测定作为 体外 模型评估内部血-视网膜屏障通透性
Summary
该协议说明了 体外 内皮细胞转吞测定作为评估内部血 – 视网膜屏障通透性的模型，通过测量人视网膜微血管内皮细胞在洞穴介导的跨细胞运输过程中跨细胞运输辣根过氧化物酶的能力。
Abstract
血液 – 视网膜屏障（BRB）的功能障碍有助于几种血管性眼病的病理生理学，通常导致视网膜水肿和随后的视力丧失。内部血视网膜屏障（iBRB）主要由生理条件下通透性较低的视网膜血管内皮组成。这种低通透性的特征受到严格调节，并通过相邻视网膜微血管内皮细胞之间的低细胞旁转运速率以及通过它们的跨细胞转运（转胞作用）来维持。视网膜跨细胞屏障通透性的评估可能为iBRB在健康和疾病中的完整性提供基本见解。在这项研究中，我们描述了一种内皮细胞（EC）转吞测定，作为使用人视网膜微血管内皮细胞（HRMECs）评估iBRB通透性的 体外 模型。该测定分别评估了HRMEC在受体和洞穴介导的跨细胞运输过程中运输转铁蛋白和辣根过氧化物酶（HRP）的能力。将培养在多孔膜上的完全融合的HRMEC与荧光标记的转铁蛋白（网格蛋白依赖性转胞作用）或HRP（洞穴介导的转胞作用）一起孵育，以测量转移到底室的转铁蛋白或HRP的水平，指示EC单层的转吞水平。Wnt信号传导是一种已知的调节iBRB的途径，被调节以证明洞穴介导的基于HRP的转吞测定方法。此处描述的EC转吞测定可为研究血管病理学中EC通透性和iBRB完整性的分子调节因子以及筛选药物递送系统提供有用的工具。
Introduction
人类视网膜是人体中能量需求最高的组织之一。神经视网膜的正常运作需要有效的氧气和营养物质供应以及其他潜在有害分子的限制通量来保护视网膜环境，这是通过血液 – 视网膜屏障（BRB）介导的1。与中枢神经系统中的血脑屏障（BBB）类似，BRB在眼睛中充当选择性屏障，调节离子，水，氨基酸和糖进出视网膜的运动。BRB还通过防止暴露于循环因子（如免疫细胞，抗体和有害病原体）来维持视网膜稳态及其免疫特权2。BRB 功能障碍有助于多种血管性眼病的病理生理学，例如糖尿病视网膜病变、年龄相关性黄斑变性 （AMD）、早产儿视网膜病变 （ROP）、视网膜静脉阻塞和葡萄膜炎，导致血管源性水肿和随后的视力丧失3，4，5。
BRB由两个独立的屏障组成，分别用于两个不同的眼血管网络：视网膜脉管系统和视网膜下方的开窗脉络膜毛细血管。内部BRB（iBRB）主要由视网膜微血管系统衬里的视网膜微血管内皮细胞（RMEC）组成，其滋养视网膜内部神经元层。另一方面，视网膜色素上皮形成外BRB的主要成分，位于神经感觉视网膜和脉络膜毛细血管之间2。对于iBRB，跨RMEC的分子转运通过细胞旁和跨细胞途径发生（图1）。iBRB的高度物质选择性取决于（i）连接蛋白复合物的存在，这些复合物限制了相邻内皮细胞（ECs）之间的细胞旁转运，以及（ii）内皮细胞内保持低跨细胞转运速率的洞穴介质，转运蛋白和受体的低表达水平1，6，7，8.调节细胞旁通量的连接复合物由紧密连接（claudins，occludins），粘附连接（VE cadherins）和间隙连接（连接蛋白）组成，允许水和小的水溶性化合物通过。虽然小的亲脂性分子被动地扩散到RMEC的内部，但较大的亲脂性和亲水性分子的运动受到ATP驱动的跨内皮途径的调节，包括囊泡运输和膜转运蛋白5，9。
水疱转胞作用可分为洞穴蛋白介导的洞穴转胞作用、网格蛋白依赖性（和受体介导的）转胞作用和网格蛋白依赖性巨胞细胞增多症（图 2）。这些囊泡转运过程涉及不同大小的囊泡，其中巨松体最大（范围为200-500nm），洞穴最小（平均为50-100nm），而网格蛋白包被的囊泡范围为70-150nm10。洞穴是带有蛋白质外壳的烧瓶形富含脂质的质膜内陷，主要由洞穴蛋白-1组成，其通过其洞穴蛋白支架结构域结合脂质膜胆固醇和其他结构和信号蛋白11。洞穴与外周附着的洞穴一起工作，以促进质膜12处的洞穴稳定。洞穴膜还可能携带其他分子的受体，如胰岛素、白蛋白和循环脂蛋白，包括高密度脂蛋白 （HDL） 和低密度脂蛋白 （LDL），以帮助它们在内皮细胞中移动13。在发育过程中，功能性BRB的形成取决于EC转吞作用的抑制8。因此，在生理条件下，成熟的视网膜内皮相对于其他内皮细胞具有相对较低的洞穴、洞穴蛋白-1 和白蛋白受体水平，这有助于其屏障特性4，9。
由于iBRB分解是许多病理性眼病的主要标志，因此开发评估体内和体外视网膜血管通透性的方法至关重要。这些方法有助于提供对BRB完整性受损机制的可能见解，并评估潜在治疗靶点的疗效。目前的体内成像或定量血管渗漏测定通常采用荧光（荧光素钠和葡聚糖）、比色法（埃文斯蓝染料和辣根过氧化物酶[HRP]底物）或放射性示踪剂14，以通过显微镜成像或分离的组织裂解物检测从脉管系统外渗到周围视网膜组织中。用于量化血管完整性的理想示踪剂应该是惰性的，并且足够大，以便在限制在健康和完整的毛细血管内时自由渗透受损血管。在活眼底荧光素血管造影（FFA）或分离视网膜平面支架中使用荧光素钠或异硫氰酸荧光素偶联葡聚糖（FITC-葡聚糖）的方法广泛用于定量体内或离体视网膜外渗。FITC-葡聚糖的优点是具有不同的分子量范围，范围为4-70 kDa，用于大小选择性研究15，16，17。FITC白蛋白（~68 kDa）是一种替代的大尺寸蛋白质示踪剂，与血管渗漏研究具有生物学相关性18。埃文斯蓝染料，心内注射19，眶后或通过尾静脉20，也依赖于其与内源性白蛋白的结合形成一个大分子，该分子可以通过大多数分光光度法检测或不太常见的荧光显微镜在平面安装20，21中进行定量。然而，这些定量或光成像方法通常不能区分细胞旁转运和经内皮转运。对于通过转吞囊泡的超微结构可视化对转吞作用进行特异性分析，通常使用诸如HRP之类的示踪分子来定位内皮细胞内的转吞囊泡，可以在电子显微镜下观察到22，23，24（图3A-C）。
开发和使用体外iBRB模型来评估内皮细胞通透性可以提供稳健和高通量的评估，以补充体内实验，并有助于研究血管渗漏的分子调节因子。评估紧密连接的细胞旁转运和完整性的常用测定包括跨内皮电阻（TEER），离子电导的量度（图4）2，25，以及使用小分子量荧光示踪剂的体外血管渗漏测定26。此外，基于转铁蛋白的转胞作用测定建模BBB已被用于探索网格蛋白依赖性转吞27。尽管如此，体外评估BRB和更具体地说，视网膜EC洞穴转吞作用的测定是有限的。
在这项研究中，我们描述了使用人视网膜微血管内皮细胞（HRMECs）作为 体外 模型的EC转吞测定，以确定iBRB通透性和EC转吞作用。该测定依赖于HRMEC分别通过受体介导或洞穴依赖性转吞途径转运转铁蛋白或HRP的能力（图2）。将培养至在顶室（即滤芯）中完全汇合的HRMEC与荧光偶联转铁蛋白（Cy3-Tf）或HRP一起孵育，以测量对应于仅通过EC转吞作用转移到底室的转铁蛋白或HRP水平的荧光强度。通过测量TEER可以确认细胞单层的汇合度，表明紧密的连接完整性25。为了证明TEER和转胞测定技术，使用了已知的血管通透性和EC转吞作用的分子调节剂，包括血管内皮生长因子（VEGF）28 和Wnt信号传导（Wnt配体：Wnt3a和Norrin）29。
Protocol
Representative Results
Discussion
BRB在视网膜健康和疾病中起着至关重要的作用。评估血管通透性的体外技术已被证明是屏障（BRB/BBB）发育和功能研究中的关键工具。这里描述的程序可用于研究EC转吞作用的分子机制或评估影响BRB通透性的相关分子调节剂。体外EC转吞测定与体内测定或用于评估血管通透性的技术相比具有多种优势。它们以高通量快速执行，可用于分析具有遗传和药理调节的特定信号通路的许?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH对JC的资助（R01 EY028100，EY024963和EY031765）。ZW得到了圣殿骑士之眼基金会职业入门补助金的支持。
Materials
| Biological Safety Cabinet | Thermo Electron Corporation, Thermo Fisher Scientific | 1286 | |
| Cell culture petridish | Nest Biotechnology | 704001 | |
| Centrifuge | Eppendorf | 5702 | |
| Centrifuge tubes (15 mL) | Corning Inc. | 352097 | |
| Centrifuge tubes (50 mL) | Denville Scientific Inc. | C1062-P | |
| Cyanine 3-human Transferrin | Jackson ImmunoResearch | AB_2337082 | |
| Endothelial Cell Basal Medium-2 (EBM-2) | Lonza Bioscience | CC-3156 | |
| Endothelial Cell Growth Medium-2 (EGM-2) SingleQuots supplements | Lonza Bioscience | CC-4176 | |
| EVOM Millicell Electrical Resistance System-2 (ERS-2) | Millipore | MERS00002 | |
| Fetal Bovine Serum (FBS) | Lonza Bioscience | CC-4102B | |
| Gelatin | Sigma-Aldrich | G7765 | |
| Hemocytometer (2-chip) | Bulldog Bio | DHC-N002 | |
| Horseradish Peroxidase (HRP) | Sigma-Aldrich | P8250 | |
| Human retinal microvascular endothelial cells (HRMEC) | Cell Systems | ACBRI 181 | |
| Incubator | Thermo Electron Corporation, Thermo Fisher Scientific | 3110 | |
| L cells (for Control-conditioned medium) | ATCC | CRL-2648 | |
| L Wnt-3A cells (for Wnt3A-conditioned medium) | ATCC | CRL-2647 | |
| Light microscope | Leica | DMi1 | |
| Multimode Plate Reader | EnSight, PerkinElmer | ||
| Phosphate-buffered saline (PBS) buffer (1x) | GIBCO | 10010-023 | |
| QuantaBlu Fluorogenic Peroxidase Substrate kit | Thermo Fisher Scientific | 15169 | |
| Recombinant human Norrin (rhNorrin) | R&D Systems | 3014-NR | |
| Recombinant human Vascular endothelial growth factor (rhVEGF) | R&D Systems | 293-VE | |
| Syringe filter (0.22 µm) | Millipore | SLGP033RS | |
| Transwell inserts (6.5 mm transwell, 0.4 µm pore polyester membrane insert) | Corning Inc. | CLS3470-48EA | |
| Trypsin-EDTA (0.25%) (1x) | GIBCO | 25-200-072 | |
| Water bath | Precision, Thermo Fisher Scientific | 51221060 | |
| XAV939 (Wnt/β-catenin Inhibitor) | Selleckchem | S1180 |
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