Department of Biological Sciences, University of North Texas, Denton, 76203, USA.
Blood Cells Mol Dis. 2010 Mar 15;44(3):169-74. doi: 10.1016/j.bcmd.2009.12.004. Epub 2010 Jan 4.
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 alphaIIb 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 alphaIIb 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 alphaIIb Vivo-Morpholino injections. The action of the alphaIIb Vivo-Morpholino was demonstrated by the presence of an alternatively spliced alphaIIb mRNA and the reduction of alphaIIb in thrombocytes of fish treated with alphaIIb 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.
通过反义寡核苷酸敲低蛋白质功能已被用于不仅在发育过程中，而且在人类疾病中理解蛋白质功能。最近，经过化学修饰的穿透细胞的 Vivo-Morpholinos 已被用于成年实验动物模型中，以改变剪接从而改变蛋白质表达。到目前为止，还没有使用 Vivo-Morpholinos 来评估成年动物的止血功能的研究。我们将 alphaIIb Vivo-Morpholinos 静脉注射到成年斑马鱼体内。通过对柠檬酸盐血液的聚集时间测定、血小板与 Annexin V 的结合以及鳃出血来测定血小板功能。alphaIIb Vivo-Morpholinos 注射后 24 小时出现血小板功能抑制，并在 48 小时达到最大值。然而，在 72 小时时，抑制不再观察到。在 alphaIIb Vivo-Morpholino 注射后 48 小时观察到 Annexin V 与血小板结合减少和鳃出血增加。alphaIIb Vivo-Morpholino 的作用通过存在另一种剪接的 alphaIIb mRNA 和 alphaIIb 在 alphaIIb Vivo-Morpholino 处理的鱼的血小板中的减少来证明。这些结果首次提供了在成年斑马鱼中可以通过血小板特异性 Vivo-Morpholinos 抑制血小板功能的原理证明，并提供了一种敲低血小板特异性基因的方法来在血小板中进行生化研究。该研究还首次提供了一种抗血栓形成的反义方法来抑制成年斑马鱼中的血小板功能。
