摘要
目的：探讨丝／苏氨酸蛋白激酶（mammaliansterile20-likekinase1，MSTl）对肿瘤坏死因子．a（tumornecrosisfactorqTNF-α）诱导的人脐静脉内皮细胞（humanumbilicalveinendothelialcell，HUVEC）凋亡的影响及作用机制。方法：用不同浓度的TNF-α（0-100ng／mE）诱导内皮细胞凋亡，24h后通过TUNEL法观察内皮细胞凋亡率，Western印迹分析TNF-α对MSTl活性的影响；随后将构建的MSTl小干扰RNA（smallinterferenceRNA，siRNA）在脂质体Lipofectamine2000的介导下转染原代HUVEC，转染后24h加入TNF-α（10ng／mL）诱导HUVEC凋亡，24h后通过Western印迹确定MSTlsiRNA的基因沉默效率；通过TUNEL法观察MSTl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MSTl基因敲除对Caspase，3活性的影响。结果：TNF-α（10，40，100ng／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，MSTl的活化增多，MSTl激酶活性相应增加；MSTlsiRNA对内皮细胞中MSTI基因表达的抑制呈剂量依赖性，100nmol／LMSTlsiRNA能特异性沉默内皮细胞中MSTl基因的表达（P〈0．05）；MSTlsiRNA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MSTl裂解活化，同时caspase-3的活性也相应减少。结论：MSTlsiRNA通过抑制caspase-3的级联放大效应减少TNF-α诱导的HUVEC凋亡。
Objective： To elucidate the effects of mammalian sterile 20-1ike 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-a for an additional 24 hours. Cell apoptosis was measured by TUNEL staining and caspase-3 activity was detected by Western blot. Results： MST 1 activity was stimulated in a dose-dependent manner after TNF-a 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 MST 1 siRNA markedly diminished MST1 gene expression in a dose-dependent manner. MST 1 siRNA （100 nmol/L） significantly silenced MST1 gene （P〈0.0S） and reduced TNF-ct-induced endothelial cells apoptosis （P〈0.0S） by way of inhibiting MST 1 gene activation and, accordingly, suppressing caspase-3 activity. Conclusion： Silencing of MST1 expression by siRNA diminishes TNF-ct-mediated human umbilical vein endothelial cell apoptosis by inhibiting the cascade effect of caspase-3.
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2012年第7期669-674,共6页
Journal of Central South University ：Medical Science
基金
国家自然科学基金(81100820)
湖南省科技厅科技计划项目(2011FJ6031)~~
