2-氯乙醇暴露对BV2细胞活化的影响  

Effect of 2-chloroethanol exposure on the activation of BV2 cells

作　　者：高怡 罗欣 杨金翰 赵凤红[1] 王高阳[1] 金亚平[1] GAO Yi;LUO Xin;YANG Jin-han;ZHAO Feng-hong;WANG Gao-yang;JIN Ya-ping(Department of Labor Health,School of Public Health,China Medical University,Shenyang,Liaoning 110122,China)

机构地区：[1]中国医科大学公共卫生学院劳动卫生教研室,辽宁沈阳110122

出　　处：《中国职业医学》2021年第6期648-654,共7页China Occupational Medicine

基　　金：国家自然科学基金(81872573)。

摘　　要：目的 探讨2-氯乙醇在BV2细胞活化中的作用与机制。方法 将对数生长期BV2细胞随机分为4组,根据细胞活力测定结果,分别采用浓度为0、40、80和160 mmol/L的2-氯乙醇刺激后,于正倒置一体显微镜下观察细胞形态;以实时荧光定量聚合酶链式反应法检测小胶质细胞活化标记物离子钙接头蛋白1(Iba1)和分化抗原11b(CD11b)mRNA的表达;以免疫印迹法检测IBA1和CD11b蛋白的相对表达水平,并检测BV2细胞的p-p65和p65在细胞核、细胞质中的表达情况;以酶联免疫吸附测定法检测细胞培养基上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-10和转化生长因子-β1(TGF-β1)水平;以免疫荧光法检测BV2细胞内p65核内移位情况。结果 与对照组比较,40、80和160 mmol/L剂量组BV2细胞呈炎症激活状态,且激活的程度随2-氯乙醇浓度增加而加重。与对照组比较,80和160 mmol/L剂量组BV2细胞中Iba1 mRNA相对表达水平均升高(P值均<0.05),160 mmol/L剂量组BV2细胞中CD11b mRNA相对表达水平升高(P<0.05),且3个剂量组BV2细胞中IBA1和CD11b蛋白相对表达水平均上调(P值均<0.05),以160 mmol/L剂量组最高(P值均<0.05)。与对照组比较,3个剂量组BV2细胞分泌的TNF-α水平呈剂量依赖性升高(P值均<0.05),IL-10水平均下降(P值均<0.05);与40 mmol/L剂量组和对照组比较,160 mmol/L剂量组BV2细胞分泌的IL-6水平均升高(P值均<0.05),TGF-β1水平均下降(P值均<0.05)。与对照组比较,80和160 mmol/L剂量组BV2细胞中p-p65蛋白相对表达水平均升高(P值均<0.05),且3个剂量组BV2细胞中细胞核p65蛋白相对表达水平均升高(P值均<0.05),细胞质中p65蛋白相对表达水平均下降(P值均<0.05),浆核比均下降(P值均<0.05)。免疫荧光检测结果显示,随着2-氯乙醇刺激浓度的增加,细胞核位置附近p65荧光强度逐渐增强。结论 2-氯乙醇刺激可导致BV2细胞形态改变,增加小胶质细胞活化标记物IBA1和CD11b的表达�Objective To explore the activation effect of 2-chloroethanol on BV2 cells and its mechanism. Methods The BV2 cells at logarithmic growth stage were randomly divided into four groups. According to the cell viability test results, the cells were stimulated with 2-chloroethanol at the concentration of 0, 40, 80 and 160 mmol/L, and the cell morphology was observed under an inverted microscope. The mRNA expression of biomarkers of activated microglia such as ionized calcium binding adaptor molecule-1(Iba1) and cluster differentiation 11 b(CD11 b) was detected by real-time fluorescence quantitative polymerase chain reaction. The Western blotting was used to detect the relative expression of IBA1 and CD11 b, as well as the expression status of the phospho Ser536(p-p65) and p65 in the nucleus and cytoplasm of BV2 cells. The levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-6 and IL-10 and transforming growth factor-β1(TGF-β1) in the supernatant of cell culture medium were determined by enzyme-linked immunosorbent assay. The translocation of p65 in BV2 cells was detected by immunofluorescence. Results In the 40, 80 and 160 mmol/L 2-chloroethanol treatment groups, BV2 cells showed an inflammatory activation state, and the activation degree increased with the increase of 2-chloroethanol concentration, compared with the control group. The relative expression of Iba1 mRNA in BV2 cells at the 80 and 160 mmol/L dose group increased(both P<0.05). The relative expression of CD11 b mRNA in BV2 cells at the 160 mmol/L dose group increased(P<0.05) compared with the control group. The relative expression of IBA1 and CD11 b protein in BV2 cells was up-regulated in the three doses groups(all P<0.05), and was the highest in the 160 mmol/L dose group(all P<0.05). Compared with control group, the level of TNF-α secreted by BV2 cell in the three doses groups increased in a dose-dependent manner(all P<0.05), and the level of IL-10 decreased(all P<0.05). The level of IL-6 secreted by BV2 cell in the 160 mmol/L dose group increa

关 键 词：1 2-二氯乙烷 2-氯乙醇 小胶质细胞 细胞活化 炎症反应 炎症因子 磷酸化 

分 类 号：R114[医药卫生—卫生毒理学] R135.1[医药卫生—公共卫生与预防医学]