摘要
目的探讨苯并(a)芘[benzo(a)pyrene,BaP]和邻苯二甲酸二丁酯(dibutyl phthalate,DBP)体外染毒对人正常肝细胞(L02)细胞信号传导与转录激活因子3(signal transducers and activators of transcription 3,STAT3)的表达及其活化(p-STAT3)的影响。方法将体外培养的L02细胞分为对照组(0.1%DMSO)、BaP低、中、高剂量单独染毒组(12.5、25.0、50.0μmol/L BaP)、DBP低、中、高剂量单独染毒组(25.0、50.0、100.0μmol/L DBP)和BaP、DBP低、中、高剂量联合染毒组(12.5μmol/L BaP+25.0μmol/L DBP、25.0μmol/L BaP+50.0μmol/L DBP、50.0μmol/L BaP+100.0μmol/L DBP)。染毒72 h后,用倒置显微镜观察细胞形态,应用实时荧光定量聚合酶链反应(reverse transcription quantitative real-time polymerase chain reaction,qRT-PCR)法检测各染毒组STAT3 mRNA的表达水平,免疫印迹(Western blot)方法观察STAT3和p-STAT3蛋白的表达水平。结果细胞染毒72 h后,12.5μmol/L BaP、25.0μmol/L DBP和12.5μmol/L BaP+25.0μmol/L DBP低剂量染毒组的细胞形态改变不明显,仅少量细胞的细胞壁模糊;25.0μmol/L BaP、50.0μmol/L DBP和25.0μmol/L BaP+50.0μmol/L DBP中剂量染毒组细胞开始出现模糊,部分细胞由不规则形变为椭圆形,细胞间隙变大;50.0μmol/L BaP、100.0μmol/L DBP和50.0μmol/L BaP+100.0μmol/L DBP高剂量染毒组贴壁细胞数量减少,漂浮细胞增多,胞体缩小变圆,细胞间隙明显变大,细胞核增大;BaP、DBP联合染毒组较其对应的单独染毒组细胞形态学改变明显。与对照组相比,除12.5μmol/L BaP、25.0μmol/L BaP、25.0μmol/L DBP和12.5μmol/L BaP+25.0μmol/L DBP组外,其余各染毒组的STAT3 mRNA表达水平皆明显降低,差异有统计学意义(P<0.05)。BaP、DBP联合染毒组中,中剂量组(25.0μmol/L BaP+50.0μmol/L DBP)的STAT3 mRNA表达水平明显低于BaP中剂量单独染毒组(P<0.05),高剂量组(50.0μmol/L BaP+100.0μmol/L DBP)的STAT3 mRNA表达水平明显低于BaP、DBP高剂量单独染毒组(P<0.05)。与对照组相比,除12.5μmol/L BaP、25.0μmol/L BaP、25.0μmol/L DBP和12.5μmol/L BaP+25.0μmol/L DBP组外,其余各染毒组STAT3、p-STAT3蛋白的表达水平皆明显降低,差异有统计学意义(P<0.05)。BaP、DBP联合染毒组中,除低剂量组(12.5μmol/L BaP+25.0μmol/L DBP)外,中(25.0μmol/L BaP+50.0μmol/L DBP)、高(50.0μmol/L BaP+100.0μmol/L DBP)剂量组的STAT3、p-STAT3蛋白的表达水平皆明显低于各BaP、DBP中、高剂量单独染毒组,差异有统计学意义(P<0.05)。结论BaP、DBP染毒可下调L02细胞的STAT3转录表达水平,抑制STAT3的活化(p-STAT3)程度,尤以BaP、DBP联合染毒时抑制作用更为明显。此可能是BaP、DBP致肝细胞凋亡损伤的重要环节。
Objective To understand the expression levels of signal transducers and activators of transcription 3(STAT3)and p-STAT3 in human normal hepatocytes(L02)exposed to benzo(a)pyrene(BaP)and dibutyl phthalate(DBP)in combination.Methods L02 cells cultured in vitro were divided into control group(0.1%DMSO),BaP low,moderate and high doses groups(12.5,25.0 and 50.0μmol/L BaP),DBP low,moderate and high doses groups(25.0,50.0 and 100.0μmol/L DBP),BaP and DBP low,moderate and high doses co-exposure groups(12.5μmol/L BaP+25.0μmol/L DBP,25.0μmol/L BaP+50.0μmol/L DBP and 50.0μmol/L BaP+100.0μmol/L DBP).After 72 hours of exposure,the morphology of cells was observed by microscope,the expression of STAT3 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR),and the protein expression levels of STAT3 and p-STAT3 were observed by Western blot.Results After 72 hours of exposure,in 12.5μmol/L BaP,25.0μmol/L DBP and 12.5μmol/L BaP+25.0μmol/L DBP groups,no obvious changes in the cells were observed,except a small number of cells with blurred cell walls.In 25.0μmol/L BaP,50.0μmol/L DBP and 25.0μmol/L BaP+50.0μmol/L DBP groups,the shape of cells became obscure and ovalized,and the intercellular spaces became larger.In 50.0μmol/L Ba P,100.0μmol/L DBP and 50.0μmol/L BaP+100.0μmol/L DBP groups,there were more floating cells and larger intercellular space,and the cells exhibited shrank and round and the nucleus became enlarged;Also,in the combined exposure group,more obvious morphological changes were found than those in the exposed groups of BaP or DBP alone.Except for 12.5μmol/L BaP,25.0μmol/L BaP,25.0μmol/L DBP and 12.5μmol/L BaP+25.0μmol/L DBP groups,the expression levels of STAT3 mRNA in the other exposed groups were significantly lower than that in the control group(P<0.05).In the groups of Ba P and DBP in combination,the expression levels of STAT3 mRNA in moderate dose group was significantly lower than that in 25.0μmol/L BaP group(P<0.05);The expression levels of STAT3 mRNA in high dose group were significantly lower than that in 50.0μmol/L BaP or 100.0μmol/L DBP groups(P<0.05).Except 12.5μmol/L BaP,25.0μmol/L BaP,25.0μmol/L DBP and 12.5μmol/L BaP+25.0μmol/L DBP groups,the expression levels of STAT3 and p-STAT3 protein in the other exposed groups were significantly lower than those in the control group(P<0.05).In the groups of BaP and DBP in conbination,except 12.5μmol/L BaP+25.0μmol/L DBP group,the expression levels of STAT3 and p-STAT3 protein in the25.0μmol/L BaP+50.0μmol/L DBP and 50.0μmol/L BaP+100.0μmol/L DBP groups were significantly lower than those in the each corresponding individual exposure group(P<0.05).Conclusions BaP and DBP can reduce the expression levels of STAT3 and inhibit the activation of STAT3(p-STAT3)in L02 cells,especially in exposure to BaP and DBP in combination,the inhibitory effect is more obvious.This may be an important link in the apoptosis of hepatocytes induced by BaP and DBP.
作者
刘益宁
陈静
马叶梅
陈文彦
岑延利
王胜利
杨光红
LIU Yi-ning;CHEN Jing;MA Ye-mei;CHEN Wen-yan;CEN Yan-li;WANG Sheng-li;YANG Guang-hong(School of Public Health,the Key Laboratory of Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,Guiyang,Guizhou 550025,China;不详)
出处
《环境与健康杂志》
CAS
北大核心
2019年第11期975-979,共5页
Journal of Environment and Health
基金
国家自然科学基金(81760578)
贵州省区域内一流学科建设项目-公共卫生与预防医学(黔教科研发[2017]85号)。