Selective labeling of zebrafish thrombocytes: Quantitation of thrombocyte function and detection during development
Selective labeling of zebrafish thrombocytes: Quantitation of thrombocyte function and detection during development
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DOI：
10.1006/bcmd.2002.0527
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发表时间：
2002-05-01
影响因子：
2.3
通讯作者：
Jagadeeswaran, P
中科院分区：
文献类型：
--
作者：
Gregory, M;Jagadeeswaran, P
斑马鱼的血小板是哺乳动物血小板的有核等同物，已经在形态学上进行了表征，但对其发育合成和生物化学的了解有限。鉴于越来越多地使用斑马鱼作为研究止血的遗传模型，分离和研究斑马鱼血栓细胞的功能是很重要的。因此，本研究的目的是分离血小板，研究其体外功能，并确定它们进入循环的发育阶段。为了实现这些目标，我们开发了一种选择性标记血小板的方法，并检测了这些细胞被已知的哺乳动物血小板激动剂激活的情况。在体外培养的全血和用亲脂染料DiI-C-18在体内标记的血液中，我们发现标记仅在单个细胞群中存在。Wright-Giemsa染色鉴定这些细胞为斑马鱼血小板。使用选择性DiI-C-18标记，我们显示了血小板激动剂对血栓细胞聚集体、丝状伪足和脂筏的反应。此外，我们还发现，通过将fitc偶联的膜联蛋白V与暴露在血栓细胞膜上的磷脂酰丝氨酸结合，可以标记聚集体。使用这些荧光标记方法，我们开发了血小板聚集的第一个微定量分析。通过这种分析，我们提供了证据，证明ADP受体P2Y(1)存在于斑马鱼的血小板中。为了研究血小板出现的发育阶段，我们将DiI-C18微量注射到斑马鱼胚胎循环中，并在受精后36小时鉴定了dii - c -18标记的血小板的存在。这些发现将有助于剖析血小板在止血中的功能，并进一步深入了解血小板在血栓形成中的作用。(C) 2002 Elsevier Science （USA）。
Zebrafish thrombocytes, the nucleated equivalents of mammalian platelets, have been characterized morphologically, but knowledge about their developmental synthesis and biochemistry is limited. Given the increasing use of zebrafish as a genetic model to study hemostasis, it is important to isolate and study the function of zebrafish thrombocytes. Therefore, the objective of this study was to isolate thrombocytes, study their function in vitro, and identify the developmental stage at which they enter circulation. To achieve these goals, we developed a method for the selective labeling of thrombocytes and assayed these cells for activation by known mammalian platelet agonists. In both in vitro incubations of whole blood and blood labeled in vivo with the lipophilic dye DiI-C-18, we found labeling in only a single population of cells. These cells were identified as zebrafish thrombocytes by Wright-Giemsa staining. Using selective DiI-C-18 labeling, we showed the formation of thrombocyte aggregates, filopodia, and lipid rafts in response to platelet agonists. Additionally, we showed that aggregates are labeled by binding FITC-conjugated annexin V to exposed phosphatidylserine on the thrombocyte membrane. Using these fluorescent-labeling methods, we developed the first microquantitative assay for thrombocyte aggregation. With this assay, we provided evidence for the presence of an ADP receptor, P2Y(1), in the zebrafish thrombocytes. To study the developmental stage at which thrombocytes appear, we microinjected DiI-C18 into the circulation of zebrafish embryos and identified the presence of DiI-C-18-labeled thrombocytes at the 36 h postfertilization stage. These findings will prove helpful in dissecting the functions of thrombocytes in hemostasis and provide further insight into the role of platelets in thrombosis. (C) 2002 Elsevier Science (USA).