Semaphorin 3A过表达对H2O2诱导的人脐静脉内皮细胞损伤的影响

摘要

目的:探讨semaphorin 3A (Sema 3A)对过氧化氢(H2O2)诱导的人脐静脉内皮细胞(HUVECs)损伤的影响.方法:构建Sema 3A过表达载体,以脂质体转染法转染HUVECs,过表达效果以qPCR和Western blot法验证;待测细胞以200 μmol/L H2O2处理4 h;qPCR法检测炎性细胞因子水平;乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)和丙二醛(MDA)水平以相应比色法检测;细胞活力以MTT法检测;流式细胞术检测细胞凋亡,凋亡相关蛋白cleaved caspase-3及Bcl-2水平以Western blot法检测.结果:Sema 3A过表达能显著增加H2O2诱导的HUVECs凋亡、炎性细胞因子分泌以及LDH和MDA含量,同时显著抑制SOD活性和细胞活力;Sema 3A对未经H2O2处理的HUVECs没有损伤效应,即其对HUVECs的损伤具有H2O2依赖性.结论:Sema 3A能显著加重H2O2诱导的HUVECs损伤,在氧化应激所致的内皮细胞损伤过程中发挥促进作用.%AIM:To explore the influences of semaphorin 3A (Sema 3A) on hydrogen peroxide (H2O2)-induced injury in human umbilical vein endothelial cells (HUVECs).METHODS:Sema 3A over-expression vectors were constructed and transfected into the HUVECs by Lipofectamine 2000, and the over-expression effect was verified by qPCR and Western blot.The HUVECs in different groups were treated with or without 200 μmol/L H2O2 for 4 h.The levels of inflammatory cytokines were measured by qPCR.The levels of lactic dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by corresponding colorimetry.The cell viability was measured by MTT assay.The cell apoptosis was analyzed by flow cytometry.The levels of apoptosis-related proteins cleaved caspase-3 and Bcl-2 were determined by Western blot.RESULTS:H2O2 induced inflammatory cytokine secretion, increased the levels of LDH and MDA, decreased SOD activity and cell viability, and increased cell apoptosis in the HUVECs.Over-expression of Sema 3A enhanced the above processes.No injury effect of Sema 3A over-expression on HUVECs without H2O2 treatment was observed, indicating that the injury effects of Sema 3A on HUVECs depended on H2O2.CONCLUSION:Sema 3A markedly enhances H2O2-induced injury in the HUVECs, which depends on H2O2.Sema 3A may promote oxidative stress-caused endothelial cell injury.