Center for Molecular Metabolism, Nanjing University of Science & Technology, Nanjing, China.
Thromb Res. 2011 Feb;127(2):122-30. doi: 10.1016/j.thromres.2010.11.025. Epub 2010 Dec 24.
Apoptotic cell death is a highly regulated genetic program, which has been observed in mature megakaryocytes fragmenting into platelets. The clock gene Per2, a key component of core clock oscillator, was involved in affecting both cell cycle control and apoptosis. Thus, loss of Per2 function may be considered potential influence of platelet formation and function.
Per2-null mice and C57BL/6 mice were used in the study. Bleeding time, platelet count, megakaryocyte count, megakaryocyte ploidy, megakaryocyte apoptosis, rate of proplatelet formation, clot retraction, platelet aggregation and secretion were performed to evaluate thrombopoiesis and hemostasis. Quantitative RT-PCR was employed to analyze genes expression in liver, bone marrow and enriched megakaryocytes.
The Per2-null mice had nearly 50% platelet counts in peripheral blood. Per2-null platelets were compromised in their ability to aggregate and secretion, consistent with a marked reduction in the number of dense and a-granules. Megakaryocytes from Per2-null mice showed no significant variation in number but increased in ploidy. Ultrastructural examination of Per2-null megakaryocytes revealed many vacuoles in demarcation membranes and reduction in platelet granules. Megakaryocytes from Per2-null bone marrow decreased the rate of proplatelet formation and impaired apoptosis. Per2-null mice showed increased both in Tpo in livers and its receptors C-mpl in bone marrow, and the megakaryocytes from these mice decreased P53 expression, consequently increased Bcl-xl and Bcl-2 level.
The clock gene Per2 modulating the apoptosis of megakaryocytes was required for platelet formation and function.
细胞凋亡是一种高度受调控的遗传程序，在成熟的巨核细胞分裂为血小板的过程中观察到了这种现象。时钟基因 Per2 是核心时钟振荡器的关键组成部分，它参与了细胞周期控制和细胞凋亡的调节。因此，Per2 功能的丧失可能会对血小板的形成和功能产生潜在影响。
本研究使用了 Per2 基因敲除小鼠和 C57BL/6 小鼠。通过测定出血时间、血小板计数、巨核细胞计数、巨核细胞倍性、巨核细胞凋亡、前血小板形成率、血栓收缩、血小板聚集和分泌，来评估血小板生成和止血功能。采用定量 RT-PCR 分析肝脏、骨髓和富集巨核细胞中的基因表达。
Per2 基因敲除小鼠外周血中的血小板计数减少了近 50%。Per2 基因敲除小鼠的血小板聚集和分泌能力受损，与致密颗粒和α-颗粒数量明显减少一致。Per2 基因敲除小鼠的巨核细胞数量没有明显变化，但倍性增加。Per2 基因敲除小鼠巨核细胞的超微结构检查显示，分隔膜中有许多空泡，血小板颗粒减少。Per2 基因敲除小鼠骨髓中的巨核细胞减少了前血小板形成的速度，并损害了细胞凋亡。Per2 基因敲除小鼠肝脏中的 Tpo 及其在骨髓中的受体 C-mpl 增加，这些小鼠的巨核细胞 P53 表达减少，进而 Bcl-xl 和 Bcl-2 水平增加。
时钟基因 Per2 调节巨核细胞的凋亡对于血小板的形成和功能是必需的。
