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（1 h, 4 h, 6 h） and at different magnitude（0.5 Pa, 1.5 Pa, 3.0 Pa） were loaded by parallel plate flow chamber system. The expression of Pim-1 gene was detected by real-time RTPCR at mRNA level and the levels of p-Akt and p-STAT3 were measured by Western Blot. The signaling inhibitors, Wortmannin（PI3 K 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 4 h after 1.5 Pa FSS. All FSS with different magnitude could increase Pim-1 expression, especially at high FSS（3.0 Pa）. Meanwhile, FSS can significantly activate the expression of p-Akt and p-STAT3. JAK inhibitor（AG490） significantly inhibited the expression of Pim-1,whereas PI3 K 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 PI3 K/Akt signaling pathway.