【摘要】 目的:探讨丝/苏氨酸蛋白激酶(mammalian sterile 20-like kinase 1,MST1)对肿瘤坏死因子-α(tumornecrosis factor-α,TNF-α)诱导的人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)凋亡的影响及作用机制。方法:用不同浓度的TNF-α(0~100 ng/mL)诱导内皮细胞凋亡,24 h后通过TUNEL法观察内皮细胞凋亡率,Western印迹分析TNF-α对MST1活性的影响;随后将构建的MST1小干扰RNA(small interference RNA,siRNA)在脂质体Lipofectamine 2000的介导下转染原代HUVEC,转染后24 h加入TNF-α(10 ng/mL)诱导HUVEC凋亡,24 h后通过Western印迹确定MST1 siRNA的基因沉默效率;通过TUNEL法观察MST1基因的敲除对TNF-α介导的内皮细胞凋亡的影响;并进一步用Western印迹观察MST1基因敲除对caspase-3活性的影响。结果:TNF-α(10,40,100 ng/mL)能导致内皮细胞凋亡显著增多(P<0.001),并且呈剂量依赖性;随着TNF-α浓度增加,MST1的活化增多,MST1激酶活性相应增加;MST1 siRNA对内皮细胞中MST1基因表达的抑制呈剂量依赖性,100 nmol/L MST1 siRNA能特异性沉默内皮细胞中MST1基因的表达(P<0.05);MST1 siRNA能明显减少TNF-α诱导的内皮细胞凋亡(P<0.05),抑制TNF-α诱导的MST1裂解活化,同时caspase-3的活性也相应减少。结论:MST1 siRNA通过抑制caspase-3的级联放大效应减少TNF-α诱导的HUVEC凋亡。更多还原

【Abstract】 Objective: To elucidate the effects of mammalian sterile 20-like kinase 1(MST1) gene on tumor necrosis factor(TNF)-α-mediated human umbilical vein endothelial cell(HUVEC) apoptosis.Methods: Cultured HUVECs were treated with either vehicle or TNF-α(1–100 ng/mL) for 24 hours.Cell apoptosis was measured by TUNEL staining,and MST1 activity was analyzed by Western blot.In order to knock down MST1 expression in HUVECs,cells were transfected with 100 nmol/L MST1 small interference RNA(siRNA) using Lipofectamine 2000 for 24 hours,and the transfection efficiency was analyzed by Western blot.MST1 siRNA-transfected cells were treated with 10 ng/mL TNF-α for an additional 24 hours.Cell apoptosis was measured by TUNEL staining and caspase-3 activity was detected by Western blot.Results: MST1 activity was stimulated in a dose-dependent manner after TNF-α treatment(10,40,100 ng/mL) and reached the maximal effect at 100 ng/mL.MST1 activity also paralleled the onset of apoptosis as determined by TUNEL staining(P<0.001).Transfection with MST1 siRNA markedly diminished MST1 gene expression in a dose-dependent manner.MST1 siRNA(100 nmol/L) significantly silenced MST1 gene(P<0.05) and reduced TNF-α-induced endothelial cells apoptosis(P<0.05) by way of inhibiting MST1 gene activation and,accordingly,suppressing caspase-3 activity.Conclusion: Silencing of MST1 expression by siRNA diminishes TNF-α-mediated human umbilical vein endothelial cell apoptosis by inhibiting the cascade effect of caspase-3.更多还原