流体剪切应力对人脐静脉内皮细胞Pim-1基因表达的影响
The effect of fluid shear stress on expression of Pim-1 in human umbilical vein endothelial cells
目的 探讨流体剪切应力对人脐静脉内皮细胞（HUVECs）中Pim-1基因表达的影响及可能的信号机制。 方法 酶消化法分离健康产妇新鲜脐静脉获取原代HUVECs，应用平行平板流动腔系统给HUVECs加载不同时间（1、4、6 h）和不同大小（0.5、1.5、3.0 Pa）的层流剪切应力，用实时定量RT-PCR检测Pim-1基因的表达水平，用蛋白质印迹法检测p-Akt和p-STAT3的表达水平，利用Wortmannin (PI3K抑制剂)、 Deguelin (Akt抑制剂) 和AG490 (JAK抑制剂) 信号阻断剂探讨信号转导途径。 结果 HUVECs在1.5 Pa 剪切应力作用4 h后Pim-1基因表达明显增加。不同强度的切应力均会刺激Pim-1基因的表达，其中3.0 Pa表达最强。切应力能显著激活p-Akt和p-STAT3的表达。AG490可以明显抑制Pim-1的表达，而Wortmannin和Deguelin增强Pim-1的表达。 结论 流体剪切应力可诱导HUVECs中Pim-1基因表达，其表达量与刺激时间和剪切应力的强度密切相关，这种作用可能通过JAK/STAT3和PI3K/Akt信号通路调节。
Objective To investigate the effect of fluid shear stress(FSS) on the expression of Pim-1 in human umbilical vein endothelial cells (HUVECs) and the molecular mechanism of Pim-1 induced by FSS. Methods HUVECs were isolated from the fresh umbilical cord of healthy mothers by 0.125% trypsin digestion. FSS under different time phase (1h, 4h, 6h) and at different magnitude (0.5Pa, 1.5Pa, 3.0Pa) were loaded by parallel plate flow chamber system. The expression of Pim-1 gene was detected by real-time RT-PCR at mRNA level and the levels of p-Akt and p-STAT3 were measured by Western Blot. The signaling inhibitors, Wortmannin (PI3K specific inhibitor), Deguelin (Akt inhibitor) and AG490 (JAK specific antagonist), were used to investigate possible signal transduction pathway. Results The expression of Pim-1 gene was significantly increased in HUVECs at 4h after 1.5Pa FSS. All FSS with different magnitude could increase Pim-1 expression, especially at high FSS(3.0Pa). Meanwhile, FSS can significantly activate the expression of p-Akt and p-STAT3. JAK inhibitor (AG490) significantly inhibited the expression of Pim-1, whereas PI3K inhibitor (Wortmannin) and Akt inhibitor (Deguelin) enhanced Pim-1 expression. Conclusions FSS can induce the expression of Pim-1 and its expression is closely related to the intensity of stimulation time and FSS. This effect may be mediated by JAK/STAT3 and PI3K/Akt signaling pathway.
Endothelial cells； Pim-1； Fluid shear stress(FSS)； Gene expression /
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国家自然科学基金项目（31670961），湖北省科技厅自然基金项目（2015CFB186）
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