乌司他丁对中性粒细胞介导的内皮细胞损伤的抑制作用

Inhibitory effect of ulinastatin on neutrophil mediated endothelial cell injury

目的研究乌司他丁对中性粒细胞介导的内皮细胞损伤的抑制作用及其机制。方法体外培养的人脐静脉内皮细胞(HUVECs)通过肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)孵育后活化,正常人血液分离的中性粒细胞经TNF-α孵育后激活,活化的HUVECs与激活的中性粒细胞共孵育建立中性粒细胞介导的内皮细胞损伤体外模型。共孵育体系中加入不同浓度的乌司他丁(100、500、1500、3000和5000 U/ml),MTT法检测内皮细胞存活率,同时检测培养液中乳酸脱氢酶活性(LDH)、一氧化氮(NO)含量和弹性蛋白酶活性。中性粒细胞与TNF-α及不同浓度的乌司他丁共孵育后,流式细胞仪检测细胞内弹性蛋白酶活性。结果激活的中性粒细胞与活化的HUVECs共孵育可以显著降低内皮细胞存活率,同时培养液中LDH活性显著升高、NO含量显著降低、弹性蛋白酶活性显著升高,乌司他丁可以剂量依赖性对抗上述变化。同时,乌司他丁可以剂量依赖性降低TNF-α激活的中性粒细胞内弹性蛋白酶的活性。结论乌司他丁对中性粒细胞介导的内皮细胞损伤的抑制作用具有剂量依赖性,其机制与乌司他丁抑制中性粒细胞内及释放到细胞外的弹性蛋白酶的活性有关。

Objective To investigate the inhibitory effect of ulinastatin on neutrophil mediated endothelial cell injury and its underlying mechanisms.Methods Human umbilical vein endothelial cells(HUVECs) were activated with TNF-α and IFN-γ in vitro.Neutrophil cells obtained from health people were separated and primed with TNF-α.In vitro neutrophil mediated endothelial cell injury model was established by activated HUVECs and primed neutrophial co-culture,serial concentrations(100,500,1500,3000 and 5000 U/ml) of ulinastatin were added to the co-culture system.Cellular viability was determined with MTT assay,lactate dehydrogenase(LDH) content,NO content and elastase activity in the culture medium were examined.Meanwhile,serial concentra-tions of ulinastatin were added to TNF-α primed neutrophial cells,and the intracellular elastase activity was measured with a flow cy-tometer.Results The co-culture process of primed neutrophil and activated HUVECs significantly decreased the viability of HUVECs,decreased the NO content and increased the LDH and elastase content in the culture medium.Ulinatatin inhibited these changes in a dose dependent manner when added in the co-culture system.Furthermore,TNF-α alone could activate the neutrophil and increased the intracellular elastase activity,and ulinastatin could decrease the intracellular elastase activity in a dose dependent manner.Conclusion Ulinastatin can dose-dependently inhibit the neutrophil mediated endothelial cell injury in an ex vivo model,this inhibitory effect is associated with the ability of ulinastatin decreasing the intracellular and extracellular elastase activity.