细颗粒物2.5在肾小管上皮细胞氧化损伤中的作用及其调控机制  

Role of particulate matter 2.5 in oxidative damage of renal tubular epithelial cells and its regulatory mechanism

作　　者：赵颖丹[1] 张天嵩[1] 马骏[1] 张伟伟[1] 易扬[1] 顾波[1] 王汉清[1] 宣怡[1] ZHAO Yingdan;ZHANG Tiansong;MA Jun;ZHANG Weiwei;YI Yang;GU Bo;WANG Hanqing;XUAN Yi(Department of Renal Medicine,Jing′an Branch Hospital,Huashan Hospital,Jing′an District Central Hospital,Shanghai 200040,China)

机构地区：[1]上海市静安区中心医院华山医院静安分院肾内科,上海200040

出　　处：《新乡医学院学报》2020年第12期1118-1125,共8页Journal of Xinxiang Medical University

基　　金：2018年静安区卫计委课题面上项目(编号:2018MS04)。

摘　　要：目的探讨细颗粒物2.5(PM 2.5)在肾小管上皮细胞HK-2细胞氧化损伤中的作用及其调控机制。方法将对数生长期的人肾小管上皮细胞HK-2细胞随机分为空白对照组、阴性对照组、PM 2.5低剂量组、PM 2.5中剂量组及PM 2.5高剂量组。空白对照组HK-2细胞正常培养,无任何处理;阴性对照组HK-2细胞进行磷酸缓冲盐溶液处理;PM 2.5低剂量组HK-2细胞给予50 mg·L-1的PM 2.5处理24 h;PM 2.5中剂量组HK-2细胞给予100 mg·L-1的PM 2.5处理24 h;PM 2.5高剂量组HK-2细胞给予200 mg·L-1的PM 2.5处理24 h。采用流式细胞术和Edu标记实验检测各组HK-2细胞凋亡率和Edu阳性细胞率,酶联免疫吸附实验法检测各组HK-2细胞氧化应激相关指标超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及总活性氧(ROS)、丙二醛(MDA)水平,实时荧光定量聚合酶链式反应检测各组HK-2细胞微RNA(miRNA)-590-3p水平变化。另取对数生长期的HK-2细胞随机分为miRNA类似物对照组、miRNA类似物组、miRNA抑制剂对照组和miRNA抑制剂组,miRNA类似物组和miRNA类似物对照组HK-2细胞分别转染miRNA mimics和miRNA mimics对照序列,miRNA抑制剂组和miRNA抑制剂对照组分别转染miRNA inhibitor和miRNA inhibitor对照序列。Targetscan数据库和双荧光素酶报告实验预测和验证miRNA-590-3p的靶点;应用Western blot检测各组HK-2细胞核因子-E2相关因子2(NFE2L2)表达水平,酶联免疫吸附实验法检测各组HK-2细胞中SOD、GSH-Px活性及ROS、MDA水平。结果PM 2.5低剂量组、PM 2.5中剂量组及PM 2.5高剂量组HK-2细胞凋亡率均显著高于空白对照组及阴性对照组(P<0.05),PM 2.5中剂量组、PM 2.5高剂量组HK-2细胞凋亡率均显著高于PM 2.5低剂量组(P<0.05),PM 2.5高剂量组HK-2细胞凋亡率显著高于PM 2.5中剂量组(P<0.05)。PM 2.5低剂量组、PM 2.5中剂量组及PM 2.5高剂量组HK-2细胞Edu阳性细胞率均显著低于空白对照组及阴性对照组,PM 2.5中剂�Objective To investigate the role of particulate matter 2.5(PM 2.5)in oxidative damage of renal tubular epithelial cell HK-2 cells and its regulatory mechanism.Methods Human renal tubular epithelial cell HK-2 cells in logarithmic growth phase were selected,and they were randomly divided into the blank control group,negative control group,low-dose PM 2.5 group,medium-dose PM 2.5 group and high-dose PM 2.5 group.The HK-2 cells in the blank control group were normal cultured without any treatment;the HK-2 cells in the negative control group were treated with phosphate buffer solution;the HK-2 cells in the low-dose PM 2.5 group were treated with PM 2.5 at a concentration of 50 mg·L-1 for 24 h;the HK-2 cells in the medium-dose PM 2.5 group were treated with PM 2.5 at a concentration of 100 mg·L-1 for 24 h;the HK-2 cells in the high-dose PM 2.5 group were treated with PM 2.5 at a concentration of 200 mg·L-1 for 24 h.The apoptosis rate and Edu positive cell rate of HK-2 cells in each group were detected by flow cytometry and Edu labeling assay.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),total reactive oxygen species(ROS)and malondialdehyde(MDA)were measured by enzyme linked immunosorbent assay.The expression of micro RNA(miRNA)-590-3p in HK-2 cells was detected by real-time fluorescent quantitative polymerase chain reaction.In addition,the HK-2 cells in logarithmic phase were selected and randomly divided into the miRNA mimics control group,miRNA mimics group,miRNA inhibitor control group and miRNA inhibitor group.The HK-2 cells in the miRNA mimics group and miRNA mimics control group were transfected with miRNA mimics and miRNA mimics control sequence,respectively.The HK-2 cells in the miRNA inhibitor group and miRNA inhibitor control group were transfected with miRNA inhibitor and miRNA inhibitor control sequence,respectively.The targets of miRNA-590-3p were predicted and verified by targetscan database and double Luciferase report experiment.The expression of nuclear factor-erythroid-2-r

关 键 词：细颗粒物2.5 肾小管上皮细胞 微RNA-590-3p 氧化损伤 核因子-E2相关因子2 

分 类 号：R692.6[医药卫生—泌尿科学]