ABSTRACT

Our previous study indicated that Clock gene could affect the thrombotic potential. In this present study, we examined the differential expression of proteins in Clock knockdown mice’s plasma, including α1-antitrypsin as a potential target. The proteins were examined in AML12 cells with Clock gene being knocked down to confirm the differential expressions. RNAs of those cells were extracted every 3 h in 24 h. The transcriptional levels of α1-antitrypsin (Serpina1a) and fibronectin (Fn1) were analyzed with the least-squares fit of a 24-h cosine function by single cosinor method, but no circadian rhythm was determined in neither of these genes. The expressions of α1-antitrypsin and fibronectin in Clock knockdown cells were found upregulated in both transcriptional and translational levels. Then, we applied ELISA assay to detect the concentration of activated protein C in Clock knockdown plasma and found a risen concentration. Fibronectin was reported to play a role in inhibiting the platelet aggregation, while activated protein C was demonstrated to inhibit PAI-1. All these results indicated that downregulation of the Clock gene in the circulatory system might have effects on PAI-1 by upregulating α1-antitrypsin, and might play some roles in platelet aggregation by increased fibronectin.

摘要

我们之前的研究表明，Clock基因会影响血栓形成的可能性。在本研究中，我们检查了时钟敲低小鼠血浆中蛋白质的差异表达，包括作为潜在靶标的 α1-抗胰蛋白酶。在敲除Clock基因的AML12 细胞中检查蛋白质以确认差异表达。在 24 小时内每 3 小时提取一次这些细胞的 RNA。α1-抗胰蛋白酶 ( Serpina1a ) 和纤连蛋白 ( Fn1 )的转录水平通过 24 小时余弦函数的最小二乘拟合通过单余弦法进行分析，但在这两个基因中均未确定昼夜节律。α1-抗胰蛋白酶和纤连蛋白的表达发现时钟敲低细胞在转录和翻译水平上均上调。然后，我们应用 ELISA 测定法检测时钟敲低血浆中活化蛋白 C 的浓度，发现浓度升高。据报道，纤连蛋白在抑制血小板聚集方面发挥作用，而活化的蛋白 C 则被证明可以抑制 PAI-1。所有这些结果表明，循环系统中Clock基因的下调可能通过上调 α1-抗胰蛋白酶对 PAI-1 产生影响，并可能通过增加的纤连蛋白在血小板聚集中发挥一些作用。