Kv1.5蛋白在内毒素致血管内皮细胞损伤中的作用机制

发布时间：2018-04-16 23:38

  本文选题：Kv.蛋白 + 人脐静脉内皮细胞　； 参考：《华中科技大学学报(医学版)》2017年04期

【摘要】：目的研究Kv1.5蛋白在内毒素致血管内皮细胞损伤中的作用机制。方法体外培养人脐静脉内皮细胞(HUVECs),给予脂多糖(LPS)刺激建立脓毒症模型,并分为:空白对照组、LPS组、LPS+Kv1.5蛋白通道特异性阻滞剂MT1(250nmol/L)组及LPS+MT2(500nmol/L)组,DAPI染色免疫荧光观察各组HUVECs凋亡情况,Western blot检测Caspase-3及Bcl-2蛋白的表达,RT-PCR检测Caspase-3及Bcl-2基因的表达。结果 DAPI染色免疫荧光显示,LPS组内皮细胞核固缩、凋亡小体形成,凋亡小体形成率由空白对照组的(4.2±0.7)%增加至(26.7±0.8)%(P0.01),而250nmol/L、500nmol/L MT预处理后,凋亡小体形成率从LPS组(26.7±0.8)%分别下降至(12.4±1.0)%、(8.5±0.9)%(均P0.01);Western blot检测结果显示,LPS组较空白对照组Bcl-2蛋白表达减少、Caspase-3蛋白表达增加(均P0.01),给予500nmol/L MT预处理后,与LPS组比较,Bcl-2蛋白表达上调、Caspase-3蛋白表达下调(均P0.01);RT-PCR检测结果显示,LPS组较空白对照组Bcl-2mRNA表达下调、Caspase-3mRNA表达上调(均P0.01),给予500nmol/L MT预处理后,与LPS组比较,Bcl-2mRNA表达上调、Caspase-3mRNA表达下调(均P0.01)。结论Kv1.5介导脓毒症相关内皮细胞损伤可能与线粒体凋亡途径有关。
[Abstract]:Objective to investigate the role of Kv1.5 protein in endotoxin-induced vascular endothelial cell injury.Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and stimulated by lipopolysaccharide (LPS) to establish septic model.It was also divided into two groups: LPS-specific blocker MT1 (250 nmol / L) and LPS MT2 (500nmol / L) group. HUVECs apoptosis was observed by immunofluorescence staining. The expression of Caspase-3 and Bcl-2 protein was detected by Western blot and the expression of Caspase-3 and Bcl-2 genes were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results the results of DAPI staining showed that the endothelial cells in lipopolysaccharide group were pyknosis and apoptotic body formation. The percentage of apoptotic bodies was increased from 4.2 卤0.7% in control group to 26.7 卤0.8% in control group. However, after pretreatment with 250 nmol / L of 500nmol / L MT, the rate of apoptotic corpuscles increased to 26.7 卤0.8%.The formation rate of apoptotic corpuscles decreased from 26.7 卤0.8% in LPS group to 12.4 卤1.0% in control group (P 0.01). The results of Western blot analysis showed that the expression of Caspase-3 protein in LPS group was significantly lower than that in control group (P 0.01), and the expression of Caspase-3 protein was increased after pretreatment with 500nmol/L MT.Compared with LPS group, the expression of Bcl 2 protein was up-regulated and down-regulated (P 0.01). The results of RT-PCR showed that the expression of Caspase-3 in lipopolysaccharide group was lower than that in control group (P 0.01). After pretreatment with 500nmol/L MT, the expression of Bcl-2 mRNA was up-regulated (P 0.01) and the expression of caspase-3 was down-regulated (P 0.01).Conclusion Kv1.5 mediated sepsis-associated endothelial cell damage may be associated with mitochondrial apoptosis.
【作者单位】： 华中科技大学同济医学院附属普爱医院重症医学科;
【基金】：武汉市卫生计生委科研基金资助项目(No.wx14c64) 湖北省自然科学基金资助项目(No.ZRY2014001335)
【分类号】：R459.7
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本文编号：1761114