摘要
目的探讨柴油机排放颗粒物(DEP)对小胶质细胞氧化应激和炎症因子表达的影响及机制。方法分离大鼠原代小胶质细胞。取大鼠原代小胶质细胞和小鼠小胶质细胞BV2,采用不同浓度的DEP(0.0、12.5、25.0、50.0 mg·L^(-1))分别处理2种细胞,分别作为对照组(0.0 mg·L^(-1)DEP)、12.5 mg·L^(-1)染毒组、25.0 mg·L^(-1)染毒组和50.0 mg·L^(-1)染毒组。采用细胞计数试剂盒-8测定2种细胞的活力;流式细胞仪检测2种细胞中活性氧(ROS)水平;Western blot法检测BV2细胞中离子钙结合适配分子1(Iba1)蛋白的表达;实时荧光定量聚合酶链式反应(qRT-PCR)法检测BV2细胞中TNF-α、IL-10 mRNA表达水平;酶联免疫吸附试验(ELISA)检测大鼠原代小胶质细胞培养液上清中TNF-α蛋白表达。取大鼠原代小胶质细胞和小鼠小胶质细胞BV2,分别分为对照组、DEP染毒组、N-乙酰半胱氨酸(NAC)处理组、NAC+DEP组;对照组和DEP染毒组细胞用达尔伯克改良伊格尔培养基(DMEM)培养,NAC处理组和NAC+DEP组细胞给予5 mmol·L^(-1)NAC预处理2~4 h;然后对照组和NAC处理组加入DMEM继续培养,DEP染毒组和NAC+DEP组加入DEP悬液(DEP终浓度为50 mg·L^(-1))进行染毒。流式细胞仪检测4组大鼠原代小胶质细胞和BV2细胞中ROS水平,qRT-PCR法检测4组BV2细胞中TNF-α、IL-10 mRNA表达水平,ELISA法检测4组大鼠原代小胶质细胞中TNF-α蛋白的表达水平。结果12.5、25.0、50.0 mg·L^(-1)染毒组原代小胶质细胞的活力显著低于对照组(P<0.05)。50.0 mg·L^(-1)染毒组BV2细胞的活力显著低于对照组(P<0.05),12.5、25.0 mg·L^(-1)染毒组BV2细胞的活力与对照组比较差异无统计学意义(P>0.05)。12.5、25.0、50.0 mg·L^(-1)染毒组原代小胶质细胞中ROS水平显著高于对照组(P<0.05)。25.0、50.0 mg·L^(-1)染毒组BV2细胞中ROS水平显著高于对照组(P<0.05),12.5 mg·L^(-1)染毒组与对照组BV2细胞中ROS水平比较差异无统计学意义(P>0.05)。12.5、25.0、
Objective To investigate the effect of diesel exhaust particle(DEP)on oxidative stress and the expression of inflammatory factors in microglia and its mechanism.Methods Primary microglia were isolated from rats.The primary microglia of rats and microglia BV2 of mouse were treated with different concentrations of DEP(0.0,12.5,25.0,50.0 mg·L^(-1))as control group,12.5 mg·L^(-1) exposure group,25.0 mg·L^(-1) exposure group and 50.0 mg·L^(-1) exposure group,respectively.The activities of cells in the two types were detected by cell counting kit-8;the levels of reactive oxygen species(ROS)in the two types cells was detected by flow cytometry;the expression of ionized calcium binding adaptor molecule 1(Iba1)protein in BV2 cells was detected by Western blot;the expression levels of TNF-αand IL-10 mRNA in BV2 cells were detected by quantitative real-time polymerase chain reaction(qRT-PCR);the expression of TNF-αprotein in the supernatant of primary microglia of rats was detected by enzyme-linked immunosorbent assay(ELISA).The primary microglia of rats and microglia BV2 of mouse were taken and divided into control group,DEP exposure group,N-acetylcysteine(NAC)intervention group and NAC+DEP group,the cells in the control group and DEP exposure group were cultured with Dulbecco′s modified Eagle′s medium(DMEM),the cells in the NAC intervention group and NAC+DEP group were pretreated with 5 mmol·L^(-1) NAC for 2-4 hours;then,the cells in the control group and NAC intervention group were added DMEM for further cultivation,the cells in the DEP exposure group and NAC+DEP group were added DEP suspension(with a final concentration of 50.0 mg·L^(-1))for exposure.The ROS levels in primary microglia of rats and BV2 cells in the above four groups were determined by flow cytometry;the expression levels of TNF-αand IL-10 mRNA in BV2 cells in the four groups were detected by qRT-PCR;the expression of TNF-αprotein in primary microglia of rats was determined by ELISA method.Results The activities of primary microglia in 12.5,
作者
张玲
王亚
徐飞
杨亦舒
韩克阳
王韶澜
刘源
杨林
安珍
李娟
吴辉
宋杰
吴卫东
ZHANG Ling;WANG Ya;XU Fei;YANG Yishu;HAN Keyang;WANG Shaolan;LIU Yuan;YANG Lin;AN Zhen;LI Juan;WU Hui;SONG Jie;WU Weidong(School of Public Health,Xinxiang Medical University,Xinxiang 453003,Henan Province,China;Nursing School,Zhenjiang College,Zhenjiang 212028,Jiangsu Province,China)
出处
《新乡医学院学报》
CAS
2023年第7期601-607,共7页
Journal of Xinxiang Medical University
基金
国家自然科学基金资助项目(编号:81961128031)。
