Vivo-Morpholino knockdown of αIIb: A novel approach to inhibit thrombocyte function in adult zebrafish
Vivo-Morpholino knockdown of αIIb: A novel approach to inhibit thrombocyte function in adult zebrafish
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DOI：
10.1016/j.bcmd.2009.12.004
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发表时间：
2010-03-15
影响因子：
2.3
通讯作者：
Jagadeeswaran, Pudur
中科院分区：
文献类型：
--
作者：
Kim, Seongcheol;Radhakrishnan, Uvaraj P.;Jagadeeswaran, Pudur
利用反义寡核苷酸敲除蛋白质的功能，不仅可以了解蛋白质在发育过程中的功能，还可以了解蛋白质在人类疾病中的功能。近年来，化学修饰的morpholinos渗透细胞，已被用于成年实验动物模型，以改变剪接从而改变蛋白质表达。到目前为止，还没有这样的研究使用活体morpholinos来评估成年动物的止血功能。我们将α IIb活体morpholinos静脉注射到成年斑马鱼体内。通过柠檬酸血的时间聚集法、膜联蛋白V与血小板的结合和鳃出血来检测血小板功能。注射α IIb Vivo-Morpholinos后24 h出现血小板功能抑制，48 h达到最大，72 h后不再观察到抑制。注射α IIb - morpholino 48小时后，观察到膜联蛋白V与血小板结合减少，鳃出血增加。α IIb活体morpholino的作用通过α IIb mRNA的选择性剪接和α IIb在经α IIb活体morpholino处理的鱼的血栓细胞中的减少得到证实。这些结果首次证明了成年斑马鱼的血小板功能可以被血小板特异性体内morpholinos抑制，并提出了一种敲低血小板特异性基因以进行血小板生化研究的方法。该研究还提供了第一个反义抗血栓方法来抑制成年斑马鱼的血栓细胞功能。(C) 2009爱思唯尔公司版权所有。
Knockdown of protein function by antisense oligonucleotides has been used to understand the protein function not only in development but also in human diseases. Recently, Vivo-Morpholinos, chemically modified morpholinos which penetrate the cells, have been used in adult experimental animal models to alter the splicing and thereby change the protein expression. Until now, there have been no such studies using Vivo-Morpholinos to evaluate hemostatic function in adult animals. We injected alpha IIb Vivo-Morpholinos intravenously into adult zebrafish. Thrombocyte function was assayed by time to aggregation assay of the citrated blood, annexin V binding to thrombocytes, and gill bleeding. The thrombocyte functional inhibition occurred in 24 h after alpha IIb Vivo-Morpholinos injection and reached a maximum in 48 h. However, in 72 h, the inhibition was no longer observed. Reduction of annexin V binding to thrombocytes and increased gill bleeding were observed 48 h after alpha IIb Vivo-Morpholino injections. The action of the alpha IIb Vivo-Morpholino was demonstrated by the presence of an alternatively spliced alpha IIb mRNA and the reduction of alpha IIb in thrombocytes of fish treated with alpha IIb Vivo-Morpholino. These results provide the first proof of principle that thrombocyte function can be inhibited by thrombocyte-specific Vivo-Morpholinos in adult zebrafish and presents an approach to knockdown thrombocyte-specific genes to conduct biochemical studies in thrombocytes. This study also provides the first antisense antithrombotic approach to inhibit thrombocyte function in adult zebrafish. (C) 2009 Elsevier Inc. All rights reserved.