黑炭与铅联合暴露致Z310细胞黏附分子表达变化

The expression of adhesion molecules in Z310 cells induced by combined exposure to black carbon and lead

目的探讨黑炭与铅联合暴露对大鼠脉络丛上皮细胞系Z310细胞中细胞黏附分子表达和调控细胞黏附分子的微小RNA(miRNA)影响。方法(1)将Z310细胞随机分为对照组、黑炭暴露组、铅暴露组和联合暴露组,铅暴露组、黑炭暴露组细胞分别予剂量为10μmol/L的乙酸铅和10 mg/L的黑炭染毒,联合暴露组予上述剂量的黑炭与乙酸铅染毒;12.0 h后,采用蛋白印迹法检测Z310细胞中细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)、黏膜血管地址素细胞黏附分子-1(MAdCAM-1)的蛋白表达,采用实时荧光定量聚合酶链反应法检测调控ICAM-1基因的miR-326、miR-328-3p和miR-542-3p的表达。(2)将Z310细胞或miRNA-326 mimic转染成功的Z310细胞随机分为对照组、miRNA-326转染对照组、联合暴露组和miRNA-326转染联合暴露组,2个对照组细胞均不予染毒处理,2个联合暴露组均予剂量为10 mg/L的黑炭染毒和10μmol/L的乙酸铅染毒12.0 h,采用蛋白印迹法检测ICAM-1的蛋白表达。结果(1)黑炭暴露组细胞中的ICAM-1、VCAM-1、MAdCAM-1和铅暴露组细胞中ICAM-1蛋白相对表达水平均高于对照组(P值均<0.05);联合暴露组细胞中ICAM-1和MAdCAM-1蛋白相对表达水平分别高于其余3组(P值均<0.05),VCAM-1蛋白相对表达水平均高于对照组和铅暴露组(P值均<0.05)。黑炭暴露组和铅暴露组细胞中miR-326相对表达水平均低于对照组(P值均<0.05),联合暴露组细胞中miR-326相对表达水平分别低于其余3组(P值均<0.05);4组细胞中miR-328-3p和miR-542-3p相对表达水平比较,差异均无统计学意义(P值均>0.05)。(2)ICAM-1蛋白相对表达水平在miR-326转染对照组细胞中低于对照组(P<0.05),在联合暴露组、miR-326转染联合暴露组细胞中均高于对照组和miR-326转染对照组(P值均<0.05),在miR-326转染联合暴露组细胞中低于联合暴露组(P<0.05)。结论黑炭与铅单独暴露均可导致Z310细胞中ICAM-1、VCAM-1、MAdCAM-1表达不同程度的上调;黑炭与铅联合暴露对ICAM-1和MadCAM-1表达上调具有协同作用,以ICAM-1变化最为显著。黑炭与铅联合暴露可能通过下调miR-326表达进而上调ICAM-1蛋白的表达。

Objective To investigate the effects of combined exposure to black carbon and lead on the expression of cell adhesion molecules and their regulating microRNAs(miRNAs) in the rat choroid plexus epithelial Z310 cells.Methods i) Z310 cells were randomly divided into control group,black carbon exposure group,lead exposure group and combined exposure group.The lead exposure group and black carbon exposure group were treated with 10 μmol/L lead acetate and 10 mg/L black carbon,respectively,and the combined exposure group was treated with both in the above doses.After 12.0 hours,the expressions of intercellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),and mucosal vascular addressin cell adhesion molecule-1(MAdCAM-1) in Z310 cells was detected by Western blotting.The expression of miR-326,miR-328-3p and miR-542-3p which regulated ICAM-1 was detected by real-time fluorescent quantitative polymerase chain reaction.ii) Z310 cells or Z310 cells transfected with miRNA-326 mimic were randomly divided into control group,miRNA-326 transfection control group,combined exposure group and miRNA-326 transfection combined exposure group.Cells in the two control groups were not treated.The two combined exposure groups were treated with 10 mg/L black carbon and 10 μmol/L lead acetate for 12.0 hours.The expression of ICAM-1 was detected by Western blotting.Results i) The relative expression of ICAM-1,VCAM-1 and MAdCAM-1 in the cells of black carbon exposure group and ICAM-1 in the lead exposure group was higher than those in the control group(all P<0.05).The relative expression of ICAM-1 and MAdCAM-1 in the combined exposure group was higher than those in the other three groups(all P<0.05).The relative expression of VCAM-1 in cells of combined exposure group was higher than those in the control group and lead exposed group(all P<0.05).The relative expression of miR-326 in cells of the lead exposure group and black carbon exposure group was lower than those in the control group(all P<0.05).The relative expression of miR-326 in the combined exposure group was lower than that in the other three groups(all P<0.05).There was no significant difference between miR-328-3p and miR-542-3p in the four groups(all P<0.05).ii) The relative expression of ICAM-1 in cells of the miR-326 transfection control group cells was lower than that in the control group(P<0.05),while in the cells in the combined exposure and miRNA-326 transfection combined exposure group,it was higher than that in the control and miRNA-326 transfection control groups(all P<0.05),and lower in the miRNA-326transfection combined exposure group than in the combined exposure group(P<0.05).Conclusion Black carbon or lead exposure can upregulate the expression of ICAM-1,VCAM-1 and MAdCAM-1 in Z310 cells.Black carbon and lead combined exposure lead to a synergistic effect on upregulation of ICAM-1 and MAdCAM-1 expression,particularly ICAM-1.The combined exposure of black carbon and lead may upregulate the expression of ICAM-1 by downregulating the expression of miR-326.