自噬参与砷诱导的人脐静脉血管内皮细胞功能失调  

Autophagy is involved in the dysfunction of vascular endothelial induced by arsenic

作　　者：王敬丘 李金玉 付晓艳 姚金印 张潇丹 张微 Wang Jingqiu;Li Jinyu;Fu Xiaoyan;Yao Jinyin;Zhang Xiaodan;Zhang Wei(Institute for Iodine Deficiency Disorders Control,Center for Endemic Disease Control,Chinese Center for Disease Control and Prevention,Harbin Medical University,Harbin 150081,China;Central Laboratory,Center for Endemic Disease Control,Chinese Center for Disease Control and Prevention,Harbin Medical University,Harbin 150081,China;Institute for Endemic Fluorosis Control,Center for Endemic Disease Control,Chinese Center for Disease Control and Prevention,Harbin Medical University,Harbin 150081,China)

机构地区：[1]哈尔滨医科大学中国疾病预防控制中心地方病控制中心碘缺乏病防治研究所,150081 [2]哈尔滨医科大学中国疾病预防控制中心地方病控制中心中心实验室,150081 [3]哈尔滨医科大学中国疾病预防控制中心地方病控制中心地氟病防治研究所,150081

出　　处：《中华地方病学杂志》2020年第4期264-268,共5页Chinese Journal of Endemiology

基　　金：国家自然科学基金(81872562)。

摘　　要：目的探讨自噬是否参与调节亚砷酸钠诱导的人脐静脉血管内皮细胞功能失调。方法分离培养人原代脐静脉内皮细胞,不同剂量亚砷酸钠处理24 h,分别为0(对照)、2、5、10、20、30、50μmol/L,CCK8法检测细胞活力。根据细胞活力结果确定后续实验染砷剂量,流式细胞术检测细胞内一氧化氮(NO)含量(亚砷酸钠处理4 h),蛋白免疫印迹法(Western blot)检测自噬标志蛋白LC3表达(亚砷酸钠处理0、6、12、24 h),电镜检测自噬体(亚砷酸钠处理12 h)。同时,以0.1 mmol/L 3-MA(自噬抑制剂)预处理人原代脐静脉内皮细胞2 h,然后30μmol/L亚砷酸钠诱导,与单独30μmol/L亚砷酸钠组比较上述检测指标。结果成功分离培养人原代脐静脉内皮细胞。与对照组[细胞活力:(99.97±5.33)%,NO含量:42048.34±789.61]比较,30μmol/L亚砷酸钠组细胞活力[(73.00±0.86)%]显著下降,细胞内NO含量(23353.97±971.85)显著降低,差异有统计学意义(P均<0.01)。30μmol/L亚砷酸钠分别处理6、12和24 h,LC3Ⅱ表达水平(5.782±2.789、9.692±2.222、5.573±2.941)明显高于处理0 h(1.000±0.383,P均<0.05),其中12 h时LC3Ⅱ表达水平最高。30μmol/L亚砷酸钠处理12 h,电镜下观察细胞内自噬体明显多于对照组。与单独30μmol/L亚砷酸钠组[细胞活力:(68.78±1.55)%;LC3Ⅱ表达水平:5.680±0.545;NO含量:13025.78±962.61]比较,3-MA预处理组细胞活力[(79.54±4.99)%]升高,LC3Ⅱ表达水平(3.956±0.398)下降,差异有统计学意义(P均<0.05);细胞内NO含量(13988.51±1671.07)差异无统计学意义(P>0.05)。结论自噬参与砷诱导的人脐静脉血管内皮细胞功能失调。Objective To explore whether autophagy is involved in dysfunction of vascular endothelial induced by sodium arsenite(NaAsO2).Methods Human primary umbilical vein endothelial cells were isolated and cultured.The cells were treated with different levels of NaAsO2[0(control),2,5,10,20,30,50μmol/L]for 24 h,and cell viability was determined using CCK8.According to the results of CCK8,the levels of arsenite used in subsequent experiments were determined,intracellular nitric oxide(NO)content(incubated by NaAsO2 for 4 h)was detected by flow cytometry,LC3 levels(incubated by NaAsO2 for 0,6,12 and 24 h)was detected using Western blotting,and autophagosome(incubated by NaAsO2 for 12 h)was observed by electron microscope.At the same time,human primary umbilical vein endothelial cells were pretreated with 0.1 mmol/L 3-MA(autophagy inhibitor)for 2 h,and induced by 30μmol/L NaAsO2,and the above detection indicators were compared with those of the 30μmol/L NaAsO2 group.Results Human primary umbilical vein endothelial cells were successfully isolated and cultured.Compared with the control group[cell viability:(99.97±5.33)%,NO content:42048.34±789.61],the cell viability[(73.00±0.86)%]and NO content(23353.97±971.85)of 30μmol/L NaAsO2 group were remarkably lower,and the differences were statistically significant(P<0.01).Incubated with 30μmol/L NaAsO2 at different time points 6,12,24 h,LC3Ⅱlevels(5.782±2.789,9.692±2.222,5.573±2.941)were significantly elevated than those of control group(1.000±0.383,P<0.05),and the LC3Ⅱlevel was the highest at 12 h.After treatment with 30μmol/L NaAsO2 for 12 h,the number of autophagosome in cells observed under electron microscope was significantly higher than that of the control group.Compared with 30μmol/L NaAsO2 group[cell viability:(68.78±1.55)%,LC3Ⅱlevel:5.680±0.545,NO content:13025.78±962.61],cell viability[(79.54±4.99)%]in 3-MA+NaAsO2 group was increased,the LC3Ⅱlevel(3.956±0.398)was decreased,and the differences were statistically significant(P<0.05);intracellular N

关 键 词：亚砷酸盐类 自噬 一氧化氮 内皮功能失调 

分 类 号：R5[医药卫生—内科学]