低浓度百草枯诱导小鼠小胶质细胞异常活化的M1/M2表型变化  

Effect of low concentration paraquat on activation of mouse microglia M1/M2 phenotypes

作　　者：李颖颖 武坷鑫 田甜 王一帆 闫伟广 黄敏 Li Yingying;Wu Kexin;Tian Tian;Wang Yifan;Yan Weiguang;Huang Min(Department of Occupational Health and Environmental Hygiene,School of Public Health and Management,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学公共卫生与管理学院职业卫生与环境卫生学系,银川750004

出　　处：《中华劳动卫生职业病杂志》2019年第12期881-887,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金项目(81560538);宁夏高等学校一流学科建设(公共卫生与预防医学学科)资助项目(NXYLXK2017B08)。

摘　　要：目的观察低浓度百草枯(PQ)对小鼠小胶质细胞(BV2)的激活情况,探讨PQ对BV2细胞M1/M2表型变化的影响。方法以BV2细胞为模型,用终浓度0、0.015、0.03、0.06、0.12、0.24、0.48μmol/L PQ及0.05μmol/L 1-甲基-4-苯基吡啶离子(MPP+)处理细胞24 h,CCK8法测定细胞存活率;用终浓度0、0.015、0.03、0.06、0.12μmol/L PQ及0.05μmol/L MPP+处理细胞24 h,酶联免疫吸附(ELISA)法测定细胞培养上清液中肿瘤坏死因-α(TNF-α)、白细胞介素(IL)-6、IL-1β含量,Transwell小室法测定细胞迁移能力,免疫荧光法和流式细胞仪测定细胞吞噬能力;用终浓度0、0.03、0.06、0.12μmol/L PQ及0.05μmol/L MPP+处理细胞24 h,蛋白免疫印迹(Western blot)法测定M1型BV2细胞标志物TNF-α、IL-6、IL-1β、一氧化氮合酶(iNOS)、CD86及M2型BV2细胞标志物Ⅰ型精氨酸酶(Arg-1)、甘露糖受体(CD206)的蛋白表达水平。结果与0μmol/L PQ组比较,0.03~0.12μmol/L PQ组BV2细胞增殖活性均明显升高,0.48μmol/L PQ组增殖活性明显抑制,差异均有统计学意义(P<0.05);0.03μmol/L PQ处理24 h的BV2细胞增殖活性明显升高(P<0.05);ELISA结果显示,与0μmol/L PQ组比较,0.03、0.06μmol/L PQ组BV2细胞上清液中TNF-α、IL-6、IL-1β含量明显升高(P<0.05);免疫荧光和流式细胞仪测定结果显示,与0μmol/L PQ组比较,0.015、0.03、0.06μmol/L PQ组细胞吞噬能力明显增强(P<0.05);Transwell结果显示,0.03、0.06、0.12μmol/L PQ组细胞侵袭能力较0μmol/L PQ组明显升高(P<0.05);Western blot结果显示,与0μmol/L PQ组比较,0.03、0.06μmol/L PQ组的M1型标志物TNF-α、IL-6、IL-1β、iNOS和CD86蛋白表达水平明显升高,0.06、0.12μmol/L PQ组M2型标志物Arg-1、CD206蛋白表达水平明显降低(P<0.05)。结论低浓度PQ可以异常激活BV2细胞,使得BV2细胞向促炎型M1型转化而抑制其向抗炎型M2型转化。Objective To observe the effect of low concentration paraquat(PQ)on activation and phenotypic M1/M2 polarization of mouse microglia cells(BV2).Methods BV2 cells were used as model,and cultured in vitro were exposed to paraquat at designed concentrations of 0,0.015,0.03,0.06,0.12,0.24,0.48μmol/L and 0.05μmol/L 1-methyl-4-phenylpyridinium(MPP+)for 24 h,and cell viability was determined by CCK8 assay.After induced by 0,0.015,0.03,0.06,0.12μmol/L PQ and 0.05μmol/L MPP+for 24 h,the contents of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and IL-1βin cell culture supernatant were determined by enzyme-linked inmunosorbent assay(ELISA).Cell migration ability was determined by transwell.Immunofluorescence(IF)and flow cytometry were used to determine the phagocytic capacity of cells.Designed concentrations of 0,0.03,0.06,0.12μmol/L PQ and 0.05μmol/L MPP+for 24 h,the protein expressions of M1 markers of BV2(TNF-α,IL-6,IL-1β,Nitric oxide synthase-iNOS,CD86)and M2 markers of BV2(Arginase type-1 Arg-1 and Mannose recepteor-CD206)were determined by Western Blot after PQ expourse(0,0.03,0.06,0.12μmol/L)and 0.05μmol/L MPP+induction.Results Compared with 0μmol/L PQ group,proliferation activity of BV2 cells was significantly increased by 0.03~0.12μmol/L PQ while inhibited by 0.48μmol/L PQ(P<0.05).The cell proliferation activity of cells treated with 0.03μmol/L PQ was significantly increased in 24 hours(P<0.05).ELISA showed that TNF-α,IL-6 and IL-1βcontents in the cell supernatant of the PQ group were significantly higher than those of 0μmol/L PQ group,especially in 0.03 and 0.06μmol/L PQ exposed group(P<0.05).The results of IF and flow cytometry showed that phagocytic capacity of 0.015,0.03 and 0.06μmol/L PQ group was significantly enhanced compared with 0μmol/L PQ group(P<0.05).Transwell showed that the cell invasion ability of 0.03,0.06,0.12μmol/L PQ was significantly higher than that of 0μmol/L PQ group(P<0.05).Western blot showed that compared with 0μmol/L PQ group,the expression levels of M1 mark

关 键 词：百草枯 小鼠 小胶质细胞 活化 表型 炎症 

分 类 号：R5[医药卫生—内科学]