活细胞成像血小板脱水和分泌流量
Summary
这项工作描述了基于荧光显微镜的方法研究血小板粘附，扩散和分泌的流动。这种多功能平台能够调查血小板功能，用于血栓形成和止血的机械研究。
Abstract
血小板是止血的重要参与者，形成血栓以密封血管性破裂。他们也参与血栓形成，形成阻塞脉管系统并损伤器官的血栓，具有威胁生命的后果。这激发了血小板功能的科学研究以及在流动条件下发生时跟踪细胞 – 生物过程的方法的发展。
血小板粘附和聚集研究的各种流动模型可用于血小板生物学的两个关键现象。这项工作描述了一种在激活期间研究流动下的实时血小板脱颗粒的方法。该方法利用与注射器 – 泵装置耦合的流动室，该注射器 – 泵装置放置在宽场倒置的基于LED的荧光显微镜下。这里描述的装置允许同时激发由荧光标记的抗体或荧光素递送的多个荧光团ent染料。在活细胞成像实验之后，可以使用静态显微镜（ 即共焦显微镜或扫描电子显微镜）进一步处理和分析覆盖玻璃。
Introduction
血小板是在血液中循环的无核细胞。它们的主要功能是在损伤部位密封血管破裂并防止失血。在这些损伤部位，内皮下胶原纤维变得暴露，随后被多聚体蛋白质von Willebrand因子（VWF）覆盖。 VWF以依赖于细胞表面1上的糖蛋白Ibα-IX-V复合物的机制循环中与血小板相互作用，降低血小板的速度。这在高剪切速率下尤其重要。随后血小板在从胶原接受活化脉冲的同时进行形态学变化。这导致不可逆的扩散，最终导致血小板聚集。这两个过程都取决于颗粒物质的分泌以促进血小板 – 血小板的串扰。其中，血小板α颗粒含有纤维蛋白原和VWF，以帮助血小板粘附和桥接血小板以整联蛋白依赖的方式结合在一起。血小板致密颗粒含有无机化合物2 ，包括钙和二磷酸腺苷（ADP），有助于增强血小板活化。此外，血小板含有（过敏）炎症3 ，补体控制蛋白4和血管生成因子5,6的介质，提出了在不同条件下这些内容是否以及如何差异释放的问题。
自20世纪80年代以来，血流动力学模型的研究对血栓形成机制的研究具有重要意义。从那时起，已经取得了很大的技术进步，并且目前开发了包括纤维蛋白形成的流动模型来测定治疗性血小板浓缩物的离体电位离体 8或研究剪切速率扰动对血栓形态的影响9 。驱动稳定粘附和生理性血栓形成（止血）与病理性血栓形成（血栓形成）的分子和细胞 – 生物学机制的差异可能非常微妙，并且促进了允许这些亚细胞的实时可视化的流动模型的发展流程。
这种设置将有价值的过程的一个例子是细胞内多磷酸盐的（再）分布和凝血因子的募集，以揭示其对纤维蛋白超微结构的时间依赖性影响10 。研究往往局限于终点分析。所描述的方法的主要目的是使得能够在流动期间血小板激活期间进行的动态亚细胞过程的实时视觉调查。
Protocol
乌特勒支大学医学中心的当地医学伦理委员会批准了用于离体研究的血液，包括本研究的血液。 解决方案准备 通过溶解10mM HEPES，0.5mM Na 2 HPO 4，145mM NaCl，5mM KCl，1mM MgSO 4和5.55mM D-吡喃半乳糖苷，制备4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）葡萄糖在蒸馏水中。 制作缓冲液的两个变体：将pH值分别设置为6.5和7.4。为每一天的实验做?…
Representative Results
图1显示了流动室的图像和实验装置;硅片的位置和尺寸;和管道连接。 图2提供了流动室的尺寸的细节。 图3和电影1显示血小板粘附和扩散在固定化VWF上的时间序列图像。 CD63是一种跨膜蛋白，其被插入静止血小板15的细胞内致密颗粒的膜中。其在血小板表面上的时间依赖性动?…
Discussion
在世界范围内，血栓形成是死亡和发病的主要原因，血小板在其发展中起着核心作用。这项工作描述了流动下血小板脱颗粒活细胞成像的方法。通常假设当血小板活化时，所有颗粒内容物直接释放到溶液中。伴随的结果表明，情况并非如此。在粘附和脱颗粒过程中，血小板保留了大量的多磷酸盐（ 图4 ）。另外，多核蛋白VWF在脱粒后与血小板表面相关（ 图5 ）。这表明控…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
CM承认国际患者组织对C1抑制剂缺乏症（HAEi），Stichting Vrienden van Het UMC Utrecht和Landsteiner输血研究基金会（LSBR）的财政支持。
Materials
| 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | VWR | 441476L | |
| Na2HPO4 | Sigma | S-0876 | |
| NaCl | Sigma | 31434 | |
| KCl | Sigma | 31248 | |
| MgSO4 | Merck KGaA | 1.05886 | |
| D-glucose | Merck KGaA | 1.04074 | |
| Prostacyclin | Cayman Chemical | 18220 | |
| Tri-sodium citrate | Merck KGaA | 1.06448 | |
| Citric acid | Merck KGaA | 1.00244 | |
| Cover glasses | Menzel-Gläser | BBAD02400500#A | 24x50mm, No. 1 = 0.13-0.16 mm thickness. |
| Chromosulfuric acid (2% CrO3) | Riedel de Haen | 07404 | CAS [65272-70-0]. |
| Von Willebrand factor (VWF) | in-house purified | ||
| Fibrinogen | Enzyme Research Laboratories | FIB3L | |
| 4 well dish, non-treated | Thermo Scientific | 267061 | |
| Human Serum Albumin Fraction V | Haem Technologies Inc. | 823022 | |
| Blood collection tubes, 9 ml, 9NC Coagulation Sodium Citrate 3.2% | Greiner Bio-One | 455322 | |
| Cell analyser | Abbott Diagnostics | CELL-DYN hematology analyzer | |
| Paraformaldehyde | Sigma | 30525-89-4 | |
| Syringe pump | Harvard Apparatus, Holliston, MA | Harvard apparatus 22 | |
| 10 mL syringe with 14.5 mm diameter | BD biosciences | 305959 | Luer-Lok syringe |
| Anti-CD63-biotin | Abcam | AB134331 | |
| Anti-CD62P-biotin | R&D Systems | Dy137 | |
| 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) | Polysciences Inc. | 9224 | |
| Streptavidin, Alexa Fluor 488 conjugate | Thermo Scientific | S11223 | |
| Immersion oil | Zeiss | 444963-0000-000 | |
| Detergent solution | Unilever, Biotex | ||
| Glycine | Sigma | 56-40-6 | |
| Polyvinyl alcohol | Sigma | 9002-89-5 | Mowiol 40-88. |
| Tris hydrochloride | Sigma | 1185-53-1 | |
| 1,4-Diazabicyclo[2.2.2]octane (DABCO) | Sigma | 280-57-9 | |
| Sheep Anti-hVWF pAb | Abcam | AB9378 | |
| Alexa fluor 488-NHS | Thermo Scientific | A20000 | |
| Glycerol | Sigma-Aldrich | 15523-1L-R | |
| Parafinn film | Bemis | PM-996 | 4 in. x 125 ft. Roll. |
| Silicone sheet non-reinforced | Nagor | NA 500-1 | 200mmx150mmx0.125mm. |
| Customized cut silicone sheet with perfusion and vacuum channels | in-house made | Made of Silicone sheet non-reinforced (Nagor, NA 500-1) | |
| 1.5 mL tubes | Eppendorf AG | T9661-1000AE | |
| Fluorescent microscope | Zeiss Observer Z1 | Equiped with LED excitation lights. | |
| Microscope software | Zeiss ZEN 2 | blue edition | |
| 18 G needle (18 G x 1 1/2") | BD biosciences | 305196 | |
| NaCl | Riedel de Haen | 31248375 | |
| Tris | Roche | 10708976 | |
| Plastic pasteur pipet | VWR | 612-1681 | 7 ml non sterile, graduated up to 3ml. |
| Silicone tubing | VWR | 228-0656 | Inner diamete. x Outer diameter x Wall thickness = 1.02 x 2.16 x 0.57 mm. |
| Microscope slides | Thermo Scientific | ABAA000001##12E | 76 x 26 x 1 mm, ground edges 45°, frosted end. |
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Citazione di questo articolo
Barendrecht, A. D., Verhoef, J. J. F., Pignatelli, S., Pasterkamp, G., Heijnen, H. F. G., Maas, C. Live-cell Imaging of Platelet Degranulation and Secretion Under Flow. J. Vis. Exp. (125), e55658, doi:10.3791/55658 (2017).