组织抑制因子-1siRNA对脓毒症血清诱导内皮细胞损伤的影响

Effect of tissue inhibitor of metalloproteinases-1 siRNA on endothelial cells injury induced by septic serum

目的探讨siRNA干扰组织抑制因子-1（TIMP-1）的表达对脓毒症血清诱导人脐静脉内皮细胞损伤的影响及其机制。方法制备脓毒症患者的血清和健康志愿者的血清，体外培养人脐静脉内皮细胞株（HUVECs），随机分为空白组（正常培养）、对照组（10％健康志愿者血清干预）、脓毒症组（10％脓毒症患者血清干预）、阴性对照组（阴性siRNA转染＋10％脓毒症患者血清干预）、TIMP-1siRNA组（TIMP-1 siRNA转染＋10％脓毒症患者血清干预）。四唑盐比色（MTT）法检测内皮细胞的存活率；酶联免疫吸附试验（ELISA）检测细胞培养上清液中基质金属蛋白酶-9（MMP-9）及TIMP-1的含量；蛋白免疫印迹（Western blot）法检测内皮细胞MMP-9、TIMP-1及血栓调节蛋白（TM）的表达。结果与对照比较，脓毒症组内皮细胞存活率于12h开始下降（P〈0．05），48h降至最低（P〈0．01），培养上清液中MMP-9及TIMP-1含量升高（P〈0．01），MMP-9及TIMP-1蛋白表达上调（P〈0．01），TM蛋白表达下调（P〈0．01）；与脓毒症组比较，TIMP-1 siRNA组内皮细胞存活率下降（P〈0．05），培养上清液中MMP-9含量升高（P〈0．05），TIMP-1含量降低（P〈0．05），MMP-9蛋白表达上调（P〈0．01），TIMP-1及TM蛋白表达下调（P〈0．01）。结论TIMP．1在脓毒症血清诱导的内皮细胞损伤中发挥保护作用。

Objective To investigate the effect and possible mechanism of tissue inhibitor of Metalloproteinases-1（TIMP-1） siRNA on human umbilical vein endothelial cells injury induced by serum of septic patient. Methods Serum samples were separately collected from septic patients and healthy controls. Human umbilical vein endothelial cells （HUVECs） were randomly divided into blank group （normal culture cells）, control group （culture medium with 10% control serum）, septic group （culture medium with 10% septic serum）, negative control group （negative siRNA ＋ 10% septic serum）, and TIMP- 1 siRNA group （TIMP-1 siRNA ＋ 10% septic serum）. The survival rate of endothelial cells was detected by MTT assay. The levels of matrix Metalloproteinase-9 （MMP-9） and TIMP-1 in supernatant of culture medium were measured by enzyme-linked immunosorbent assay （ELISA）. The levels of MMP-9, TIMP- 1 and Thrombomoduline （TM） in endothelial cells were examined by Western blot. Results Compared with control group, the cell survival rate of septic group decreased 12 hours after the addition of serum （P〈0.05） and reached minimum 48 hours later. The levels of MMP-9 and TIMP-1 in supernatant of culture medium of septic group significantly increased （P〈0.01）. The levels of MMP-9 and TIMP-1 increased in the septic group （P〈0.01）, while the level of TM reduced at the same time in septic group （P〈0.01）.Compared with septic group, the cell survival rate of TIMP-1 siRNA group decreased （P〈0.05）. The level of MMP-9 in supernatant of culture medium of TIMP-1 siRNA group increased （P〈0.05）, while the level of TIMP-1 decreased （P〈0.05）. The level of MMP-9 increased in TIMP-1 siRNA group （P〈0.01）, whereas the levels of TIMP-1 and TM reduced in TIMP-1 siRNA group （P〈0.01）. Conclusions TIMP-1 plays a protective role in endothelial cells injury induced by septic serum.