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Kv1.5蛋白与脂多糖诱导的血管内皮细胞损伤的相关性

The effects of Kv1. 5 on sepsis - induced endothelial cell injury
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摘要 目的探讨Kv1.5蛋白与脂多糖(LPS)诱导的血管内皮细胞损伤的相关性。方法LPS以浓度梯度(0.5、1.0、5.0、10.0 μg/ml)及时间梯度(0、6、12、24、48 h)作用于人脐静脉内皮细胞(HUVECs),应用流式细胞仪检测细胞凋亡率,建立内皮细胞损伤模型,并通过Western blot法检测内皮细胞中Kv1.5蛋白水平;分别予以75、125、250、500 mmol/L浓度的Kv1.5特异性通道阻滞剂(MT)预处理30 min阻断Kv1.5通道后,运用流式细胞术检测内皮细胞凋亡率,酶联免疫吸附试验(ELISA)法测定内皮细胞损伤标志物内皮细胞特异性分子-1(endocan)、血管细胞间黏附分子-1(VCAM-1)的浓度。结果LPS浓度为0.5、1.0、5.0、10.0 μg/ml刺激HUVECs 24 h,内皮细胞凋亡率分别为(33.530±1.266)%、(35.530±0.551)%、(48.270±0.901)%、(51.600±2.179)%,与对照组比较差异有统计学意义(P值分别为0.025、0.027、0.011、0.004),且在LPS 5.0 μg/ml时作用最显著;LPS(5.0 μg/ml)处理内皮细胞6、12、24、48 h,内皮细胞凋亡率分别为(30.200±1.113)%、(32.470±0.814)%、(46.630±0.513)%、(50.870±1.498)%,与对照组比较差异有统计学意义(P值分别为0.027、0.025、0.012、0.006),并且在24 h时作用最明显;建立内皮细胞损伤模型适宜的条件为5.0 μg/ml LPS刺激HUVECs 24 h;同时可见不同浓度LPS组Kv1.5蛋白表达明显上调(P值分别为0.036、0.027、0.008、0.005),12、24、48 h时LPS组Kv1.5蛋白表达亦上升(P值分别为0.004、0.036、0.007),Kv1.5蛋白表达与LPS呈一定的浓度及时间依赖性。与LPS组比较,不同浓度梯度MT组(75、125、250、500 mmol/L)内皮细胞凋亡率明显降低(P值均为0.000),且细胞损伤标志物endocan(P值分别为0.001、0.000、0.001、0.006)及VCAM-1分泌减少(P值分别为0.006、0.000、0.000、0.000)。结论内毒素诱导损伤的血管内皮细胞表达Kv1.5蛋 ObjectiveTo investigate the correlation between Kv1.5 protein and lipopolysaccharide (LPS)-induced endothelial cell injury.MethodsLPS effected on human umbilical vein endothelial cells (HUVECs) in a concentration gradient (0.5, 1.0, 5.0, 10.0 μg/ml)and time gradient (0, 6, 12, 24, 48 h), then measure the cell apoptosis rate by flow cytometry to establish the model of endothelial cell injury, and detect the level of endothelial cells Kv1.5 protein by Western blotting; different concentrations of mephetyl tetrazole (MT, Kv1.5-specific channel blockers) (75, 125, 250, 500 mmol/L) was used to pretreatment 30 min to block Kv1.5 channels, then used flow cytometry to evaluate endothelial cell apoptosis rate, Enzyme linked immunosorbent assay (ELISA) assay to detect the concentrations of endothelial cell injury markers endocan and VCAM-1.ResultsIncubated HUVECs with 0.5, 1, 5, 10 μg/mlfor 24 h, the rates of Endothelial cells apoptosis were (33.530±1.266)%, (35.530±0.551)%, (48.270±0.901)%, (51.600±2.179)%, it is statistical different compared with control group (the values of P were 0.025, 0.027, 0.011, 0.004). Then treated HUVECs for 6, 12, 24, 48 h, LPS could induce HUVECs endothelial cell injury, the rates of Endothelial cells apoptosis were (30.200±1.113)%, (32.470±0.814)%, (46.630±0.513)%, (50.870±1.498)% (the values of P were 0.027, 0.025, 0.012, 0.006). Respectively, the rate of Endothelial cells apoptosis was obvious when treated with 5 μg/ml LPS for 24 h. Endothelial cells apoptosis was induced by LPS in a concentration and time-dependent, endothelial cell injury model was 5 μg/ml LPS treatment for 24 h. Kv1.5 protein expression in all groups treated with LPS of different concentration was significantly increased (P=0.036, 0.027, 0.008, 0.005) in LPS group(12, 24、48 h) (the values of P were 0.004, 0.036, 0.007), the expression of Kv1.5 protein increased in a dose and time-dependent partly. Compared with LPS group, endot
作者 许关霞 崔乐 王必蓉 余和平 许涛 Xu Meixia , Cui Le , Wang Birong ,Yu Heping, Xu Tao( Department of Surgical Intensive Care Unit, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China ; Department of General Surgery, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China )
出处 《中华实验外科杂志》 CSCD 北大核心 2017年第5期755-757,共3页 Chinese Journal of Experimental Surgery
关键词 KV1.5 人脐静脉内皮细胞 Kv1.5通道特异性阻滞剂 血管内皮细胞损伤 Kv1. 5 Human umbilical vein endothelial cells Mephetyl tetrazole Endotheli-al cell injury
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