三苯氧胺诱导血小板凋亡的研究

Study of tamoxifen induces blood platelet apoptosis

目的探讨三苯氧胺(TAM)诱导血小板凋亡作用及其分子机制。方法取健康志愿者静脉血,分离得到富含血小板血浆(PRP)和洗涤血小板,血小板与不同浓度TAM孵育后,流式细胞仪检测磷脂酰丝氨酸(PS)暴露、线粒体跨膜电位(ΔΨm)去极化、胞质Ca2+浓度;Western blot检测活化的caspase-3和gelsolin酶解产物。在抑制实验中,血小板先与线粒体通透性转换孔(MPTP)抑制剂环孢素-A(Cyp-A)或二甲基亚砜(DMSO)预孵育,然后再与TAM孵育,流式细胞仪和Western blot检测凋亡标志物。结果 TAM浓度依赖地诱导血小板PS暴露、ΔΨm去极化、胞质Ca2+浓度增加和活化的caspase-3。Cyp-A抑制TAM诱导的PS暴露、ΔΨm去极化和caspase-3活化,但不抑制胞质Ca2+浓度升高。结论 TAM能够诱导血小板发生凋亡。

Objective To investigate the effects of tamoxifen（TAM） on blood platelets and explore its possible mechanism.Methods Washed platelets were prepared from fresh blood,and blood then incubated with various concentrations of TAM.Depolarization of mitochondrial inner transmembrane potential（ΔΨm）,PS exposure and cytosolic Ca2＋ levels were analyzed by flow cytometry.Caspase-3 activation and gelsolin cleavage were detected by Western blot.To evaluate the effect of cyclosporine A（Cyp-A）,an inhibitor of mitochondrial permeability transition pore（MPTP）,on TAM-induced platelet apoptosis,washed platelets were pre-incubated with Cyp-A or DMSO before treatment of TAM.Results TAM could dose-dependently induce depolarization of ΔΨm,PS exposure,caspase-3 activation,and elevation of cytosolic Ca2＋ levels.Cyp-A inhibited TAM-induced depolarization of ΔΨm,PS exposure,and caspase-3 activation,but did not inhibit elevation of cytosolic Ca2＋ levels.Conclusion TAM induces platelet apoptosis,and MPTP may play an important role in TAM-induced platelet apoptosis.