Staurosporine对尼古丁诱导人脐静脉内皮细胞表达t-PA与PAI-1的影响

Effects of Staurosporine on nicotine induced expression of t-PA and PAI-1 in human umhilical vein endothelial cells

目的通过观察不同浓度Staurosporine（STS）对尼古丁诱导人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）表达组织型纤溶酶原激活物（tissue type plasminogen activator, t-PA）与1型纤溶酶原激活物抑制剂（plasminogen activator inhibitor-1，PAI-1）mRNA及蛋白的影响，探讨尼古丁所致内皮细胞纤溶紊乱的机制。方法将体外培养的3～6代HUVECs随机分为对照组、尼古丁组及不同浓度STS组，STS组分别以20、50、100μmol／L STS预处理细胞30min，再与100μmol／L尼古丁孵育24h。酶联免疫吸附双抗体夹心法检测细胞上清液tPA与PAI-1蛋白含量，RTPCR检测PAI-1mRNA表达。结果尼古丁组PAI-1mRNA与蛋白表达较对照组升高（P值均〈0．05）；不同浓度STS组PAI1mRNA与蛋白表达较尼古丁组降低（P值均〈0．05），且呈浓度依赖性，以100μmol／LSTS组作用为著，但PAl1mRNA与蛋白表达仍高于对照组（P值均〈0．05）。各组间t-PA蛋白差异无统计学意义（P值均〉0．05）。结论STS通过阻断蛋白激酶C通路的信号传导可部分减弱尼古丁诱导的血管内皮细胞纤溶功能紊乱。

Objective To detect the mechanism of the effects of different concentrations of Staurosporine on nicotine induced expression of tissue type plasminogen activator （t-PA） and plasminogen activator inhibitor-1 （PALl） at mRNA and protein levels in human umbilical vein endothelial cells （HUVECs）. Methods Cultured HUVECs at passage 3 to 6 were divided into the control group,nicotine group,various-dose Staurosporine groups. After intervemed by different concentrations （20,50,100 μmol/L） of Staurosporine for .30 minutes, the various-dose Staurosporine groups were stimulated by nicotine （100μmol/L） for 24 hours. The protein levels of t-PA and PAI-1 in the cultured medium were measured by enzyme linked immunosorbent assay. PAI-1 mRNA level was assayed by reverse transeript-polymerase chain reaction. Results Compared to control group, the expressions of PAI-1 mRNA and protein increased in nicotine group （all P 〈0.05）. Compared to nicotine group, the expressions of PAI-1 mRNA and protein in various-dose Staurosporine groups decreased by means of dose-dependent, the effect of 100 μmol/l. Staurosporine was notable, but the expressions of PAI-1 mRNA and protein were higher than those in control group （all P 〈0.05）. However, the expression of t PA antigens had no significant change among all groups （all P 〈0.05）. Conclusions Through inhibiting the activation of PKC signaling pathway,Staurosporine partly improves the fibrinolytic function of HUVECs induced by nicotine in vitro.