尼古丁对血管内皮细胞自噬的影响
投稿时间:2015-11-26  修订日期:2015-12-14  点此下载全文
引用本文:马琴,郭蕊,闫喆一,崔桂丽,闫佳敏,王亚静.尼古丁对血管内皮细胞自噬的影响[J].医学研究杂志,2016,45(7):131-134
DOI: 10.11969/j.issn.1673-548X.2016.07.035
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作者单位E-mail
马琴 030001 太原, 山西医科大学生理学系  
郭蕊 030001 太原, 山西医科大学生理学系  
闫喆一 030001 太原, 山西医科大学生理学系  
崔桂丽 030001 太原, 山西医科大学生理学系  
闫佳敏 030001 太原, 山西医科大学生理学系  
王亚静 030001 太原, 山西医科大学生理学系 yajinglove_yj@163.com 
中文摘要:目的 初步探讨尼古丁对人脐静脉内皮细胞(human umbilical vein endotheliel cell,HUVEC)自噬的影响。方法 采用蛋白免疫印迹(Western blot)技术分别检测尼古丁不同浓度(0、6×10-8、6×10-7、6×10-6和6×10-5mol/L)和不同时间(浓度为6×10-6mol/L时,分别干预0、1.5、3、6和12h)作用后,人脐静脉内皮细胞中自噬标志性蛋白微管相关蛋白轻链3Ⅱ(LC3Ⅱ)蛋白表达水平的变化,以确定尼古丁的最佳刺激浓度和最佳刺激时间。依据尼古丁最佳条件将体外培养的HUVE细胞随机分组:对照组、尼古丁处理组,Western blot法检测LC3Ⅱ、多聚泛素结合蛋白p62/SQSTM1(p62)和溶酶体相关膜蛋白-2(LAMP-2)的蛋白表达,Annexin Ⅴ-FITC/PI双染流式细胞术(flow cytometry,FCM)检测细胞凋亡率。结果 体外实验显示,LC3Ⅱ蛋白表达呈浓度和时间依赖性,且尼古丁浓度为6×10-6mol/L、时间为6h时,LC3Ⅱ蛋白表达最多(P<0.01)。与对照组相比,尼古丁处理组LC3Ⅱ、p62蛋白表达显著增加(P<0.05),LAMP2蛋白表达减少(P<0.05),细胞凋亡率升高(P<0.01)。结论 尼古丁可以引起自噬体与溶酶体融合障碍,使自噬降解异常,导致内皮细胞自噬功能紊乱,促进细胞凋亡。
中文关键词:尼古丁|人脐静脉内皮细胞|自噬|LC3Ⅱ
 
Effects of Nicotine on Utophagy in Vascular Endothelial Cells
Abstract:Objective To explore the influence of nicotine on autophagy in human umbilical vein endotheliel cells(HUVECs). Methods HUVECs were firstly treated with different concentrations of nicotine(0,6×10-8,6×10-7,6×10-6 and 6×10-5μmol/L).The expression of specific protein marker in autophagy mictotubule-associated protein light chain 3Ⅱ(LC3Ⅱ) in each group was detected with western blot and the highest level of LC3Ⅱ protein indicated the optimal stimulating concentration.Then, HUVECs were treated with nicotine for different times(0,1.5,3,6 and 12h) in their optimum concentration 6×10-6μmol/L. A similar western blot detection was performed in order to determine the optimal time of nicotine.Divided HUVECs cultured in vitro randomly based on the optimum condition for nicotine:control group, nicotine treatment group. The expression of LC3Ⅱ, ubiquitin binding protein p62/SQSTM1 (p62) and lysosome membrane protein-2 (LAMP-2) protein were detected by western blotting, cell apoptosis was detected by flow cytometry (FCM)using Annexin Ⅴ-FITC/PI double labeled. Results The expression of LC3Ⅱ protein was dose and time dependent.It expressed most(P<0.05) with nicotine at the time of 6h and concentration of 6×10-6mol/L. Compared with control group, nicotine treatment group had a higher level of LC3Ⅱ and p62 protein. However, the expression of LAMP-2 was decreased at the same time. Conclusion Nicotine impaires the fusion of autophagosomes and lysosomes,leading to autophagicclearance impairment and leading to endothelial cell autophagy function disorder.This may makes contribution to the cell apoptosis.
keywords:Nicotine|Human umbilical vein endotheliel cell|Autophagy|LC3Ⅱ
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